Method of enhancing cellular production of molecular chaperone, hydroxylamine derivatives useful for enhancing the chaperone production and the preparation thereof

ABSTRACT

A method of increasing expression of a molecular chaperon by a cell and/or enhancing the activity of a molecular chaperon in cells is provided. The method comprises treating a cell that is exposed to a physiological stress which induces expression of a molecular chaperon by the cell with an effective amount of a certain hydroxylamine derivative to increase the stress. Alternatively, an hydroxylamine derivative can be administrated to a cell before it is exposed to a physiological stress which induces expression of a molecular chaperon by the cell. Preferably, the cell to which an hydroxylamine derivative is administered is an eukaryotic cell. The hydroxylamine derivative corresponds to the formulae (I) or (II). 
     The invention also provides novel hydroxylamine derivatives falling within the scope of the formulae (I) and (II) as well as pharmaceutical and/or cosmetical compositions comprising the said compounds.

CROSS REFERENCE TO RELATED APPLICATION

This application is a division of U.S. patent application Ser. No.08/860,582, filed Dec. 7, 1999, which bases priority from PCTApplication No. PCT/HU96/00064, filed Nov. 1, 1996, which claimspriority from Hungarian Patent Application No. P 95 03141, filed Nov. 2,1995, Hungarian Patent Application No. P 95 03141/3919, filed Feb. 9,1996, and Hungarian Patent Application No. P 95 03141/29820, filed Oct.4, 1996.

BACKGROUND OF THE INVENTION

Molecular chaperons are proteins which mediate protein folding. Theybind non-covalently to exposed surfaces of proteins that are newlysynthesized or are denatured or misfolded, and assist them to fold intocorrect conformations. Molecular chaperons are also involved in a numberof cellular processes such as protein synthesis, protein translocationand DNA replication.

Molecular chaperons include heat shock proteins, which are proteinswhose expression increases significantly in cells following an exposureto unusually high temperature (heat shock) or an exposure to a widevariety of physiological stresses. This increase in the molecularchaperon expression in turn provides cells with protection against theadverse effects of hyperthermia, as demonstrated by the thermotoleranceof cells for otherwise lethal temperatures if the cells arepre-conditioned by a brief exposure to high temperature.

Physiological stresses inducing heat shock protein expression include awide variety of pathological conditions associated with many diseases.The synthesis of heat shock proteins in cells exposed to such stresses,indicates the protection of the cell against the physiological stresses,like also in the case of the heat shock response.

One such pathological condition associated with induction of molecularchaperons is ischemic injury. Ischemic injury to tissues results fromdeterioration of blood supply for any possible. For instance, prolongedcoronary occlusion causes severe damage to myocardium, leading tomyocardial necrosis and jeopardizing the chances for recovery even ifthe blood flow is restored. In brain, to significant damages mayfrequently be caused by ischemia, leading to death of the brain-tissue.

It was observed that the amount of heat shock protein hsp70 increased inthe myocardium during ischemia leading to necrosis even if the durationof ischemia is short. In these cases, likewise in a heat shock, theenhanced hsp70 content of the cells protects the same against theconsequences of a next ischemia, which would otherwise cause necrosis(DAS, D. K., et al. Cardiovascular Res.: 578, 1993). It has also beenobserved when rat cells in culture were subjected to ischcmia, J. Clin.Invest., 93: 759-767 (1994)). Accordingly, heat shock proteinssynthesized by myocardial cells provide protection against ischemicinjuries.

The situation in brain-tissue is similar, wherein cerebral ischemiaresults in increased expression of heat shock protein in the braintissue. Experiments have also proved that pretreatment of animals withsub-lethal ischemia induces heat stress protein (hsp70) and protects thebrain against more severe subsequent ischemic insult. (Simon, et al.,Neurosci. Lett., 163:135-137 (1993)).

Yet another example of physiological stress on tissues and organsassociated with molecular chaperon induction is provided by inflammatorydiseases. Inflammation is a non-specific response of host cells to entryof foreign material such as in case of infection by various bacterialand viral pathogens, and involves aggregation and activation ofleukocytes to the injury site, which results in production and releaseof high levels of reactive oxygen species and cytokines. These cytokinesand reactive oxygen radicals attack the pathogen, but also damage thehost tissues (Jaquier Sarlin, Experientia, 50: 1031-1038/1994/). It isbelieved that as a protection against these toxic mediators ofinflammation, the host tissues increase production of molecularchaperons. Molecular chaperons thus produced protect host cells fromdamages caused by reactive oxygen species and protect cells fromcytotoxicity of TNF and other cytokines and reactive oxygen radicals. Inanimal studies, it has been demonstrated the pre-exposure of an animalto heat shock, with resulting increase of a heat shock protein (hsp70)expression, resulted in remarkable decrease in pulmonary inflammation.Accordingly, molecular chaperons serve anti-inflammatory function.

The above examples illustrate ability of molecular chaperons to protectcells against various physiological stresses disturbing cellularhomeostatic balance and causing injury to cells. Molecular chaperonshave also been shown to be advantageous in treating neoplasms. Forexample, it has been reported that when tumor cells are transfixed witha gene encoding a molecular chaperon (65 kd hsp), they lose or showdecrease in their tumorigenicity (PCT Application No. PCT/GB93/02339).Furthermore, it has also been reported that tumor cells, in response toheat stress, express molecular chaperons in increased amount. However,they are present not in cyto-plasm, but on the surface of cellmembranes. (Ferrarini, M. et al Int. J. Cancer, 51:613-619/1992/).Increased presence of molecular chaperones on cell surfaces correlateswith increased sensitivity of NK (natural killer) cells toward the tumorcells, allowing better targeting, infiltrating, and killing of the tumorcells by NK cells (Kurosawa. S. et al. Eur. J. Immunol. 23:1029/1993/).

In view of the advantages associated with increased molecular chaperonexpression in cells, a method which increased such expression orincreased activity of molecular chaperons would be highly desirable.

SUMMARY OF THE INVENTION

The invention relates to methods for increasing expression or enhancingactivity of molecular chaperons by a cell. In particular, according toone non-limiting embodiment of the invention, a method is providedcomprising treating a cell that is exposed to a physiological stresswith an effective amount of a chemical compound during, before or afterthe physiological stress which increases expression of a molecularchaperon in the cell beyond the amount induced by the physiologicalstress,

wherein the chemical compound is a hydroxylamine derivative thetautomeric forms of which are represented by formulae (I) and (II), orits salt, including the optically active stereoisomers thereof, wherein

A is an alkyl, substituted alkyl, aralkyl, aralkyl substituted in thearyl and/or in the alkyl moiety, aryl, substituted aryl, heteroaryl orsubstituted heteroaryl group,

Z is a covalent bond, oxygen or ═NR³ wherein R³ is selected from thegroup consisting of hydrogen, an alkyl, substituted alkyl, aryl,substituted aryl, aralkyl, or aralkyl substituted in the aryl and/or inthe alkyl moiety,

R is an alkyl or substituted alkyl,

X in the tautomer of formula (I) is halogen or a substituted hydroxy oramino, monosubstituted amino or disubstituted amino group and

X in the tautomer of formula (II) is oxygen, imino or substituted iminogroup and

R′ is hydrogen, an alkyl, substituted alkyl, aryl, substituted aryl,aralkyl, aralkyl having substituted aryl and/dr alkyl moiety, acyl orsubstituted acyl group,

and the compounds of formula (I) optionally contain intramolecular ringstructures formed by coupling X and a reactive substituent.

An other non-limiting embodiment of the invention is the method ofenhancing the activity of a molecular chaperon in a cell exposed to aphysiological stress which comprises administering an effective amountof a hydroxylamine derivative of structure (I) or (II), as describedabove. Thus, the activity of molecular chaperon is increased beyond theamount induced by the physiological stress alone. In either of thesemethods, it is preferred that the cell to which the hydroxylaminederivative is administered to is an eukaryotic cell.

According to the invention eucaryotic cells are treated with thehydroxylamine derivatives as defined above.

Another object of the invention is the method of treatment or possibleprevention of diseases connected with functioning of the chaperon systemor associated with damages of the cell- or cell-organellum membrane,wherein for suppressing the pathological condition effective amount of ahydroxylamine derivative of the formula (I) or (II) is administered tothe host organism.

Still another object of the invention is the use of the hydroxylaminederivatives of the formula (I) or (II) or the salts thereof in thepreparation of pharmaceutical compositions which can be used in thetreatment of cardiovascular, vascular, cerebral; tumorous diseases,diseases of the skin and/or mucous membrane or those of the epithelialcells of renal tubules, as well as in the preparation of cosmeticalcompositions.

The invention further relates to novel hydroxylamine derivativespossessing a wide range of biological effect and are useful forenhancing the level of molecular chaperon in organisms or the activityof the said molecular chaperons and for the preparation ofpharmaceutical and cosmetical compositions applicable to this purpose.

A further object of the invention is represented by the pharmaceuticaland cosmetical compositions which comprise novel hydroxylaminederivatives together with carriers and auxiliaries generally acceptablein such compositions.

The present invention is based, at least in part, on an unexpecteddiscovery that hydroxylamine derivatives having structures as describedabove, when used in the treatment of cells, are capable of increasingthe amount of molecular chaperons produced by that cell or enhancing theactivity thereof. This effect is particularly great when the cell isunder physiological stress which induces molecular chaperon expression.In such cases, the chemical compound enhances expression of molecularchaperons by the cell beyond that amount induced by the physiologicalstress alone. This discovery is significant in view of the rolemolecular chaperons play in cells defending themselves againstpathological effects of various diseases. Thus, if a compound is able toincrease the amount or enhance the activity of molecular chaperons beingexpressed by cells, this allows the cells to be protected against thedeleterious effects of the diseases and to repair damages caused bythem.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the changes in hsp level on H9c2 rat myocardium exposed toheat shock by the effect of treatment withN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridinecarboximidoyl chloridemaleate. This compound is labeled B on the Figures and referred to ascompound B in the followings as well.

FIG. 2 shows the results of the above experiment obtained by Westernblot analysis based on densitometric evaluation.

FIG. 3 shows the results of hsp70 mRNA Northern blot analysis obtainedduring examination of the effect of compound B on cellular hspexpression at transcription level.

FIG. 4 shows the results of hsp26 mRNA Northern blot analysis obtainedon Saccharomyces cerevisiae cells during examination of the effect ofcompound B on hsp activation.

FIG. 5 shows the effect of benzyl alcohol on the adenylate cyclateactivation and the membrane state of plasma.

FIG. 6 shows the hsp gene expression rate on HeLa cells, usingluciferase reporter gene for the test.

FIG. 7 illustrates the effect of compound B on hsp72 cell surfaceexpression in K562 cell line.

FIG. 8 shows the interaction of compound B and different lipid membranesshowing the increase of surface pressure.

FIG. 9 shows the effect of compound B in the concentration of 10 mM and100 mM on the bilayer (L_(a))=T hexagonal (H₁₁) phase transfer of largeunilamellar vesicules prepared from dipalmitoyl-phosphatidylethanolamine.

FIG. 10 is the diagram of the effect of compound B on the serum TNFlevel in healthy and STZ diabetic rats.

FIG. 11 shows the effect of compound B against the growth inhibitingeffect of keratinocyte cyclohexylimide.

FIG. 12 shows the effect of compound B against the cell damaging effectof cycloheximide on endothelial cells.

FIG. 13 shows the cytoprotective effect of compound B against the celldamaging effect of cycloheximide on HeLa cell line.

FIG. 14 shows the effect of compound B against the growth inhibitingeffect of cyclohexylimide on H9c2 rat myocardium cell line.

FIG. 15 shows the effect of compound B on the PI transcription factoractivity in AB 1380 yeast cells.

FIG. 16 shows the effect of compound B on the AP 1 transcription factoractivity in JF1 yeast cells.

FIG. 17 shows the effect of compound B on the P1 transcription factoractivity in AB 1380 yeast cells.

FIG. 18 shows the test results obtained on isolated functioning ischemicrat heart model wherein the model was treated with compound B,determined by Western blotting 2 hours after ischemia.

FIG. 19 shows the test results obtained on isolated functioning ischemicrat heart model wherein the model was treated with compound B,determined by Western blotting 3 hours after ischemia.

FIG. 20 shows the wound healing on STZ diabetic rats after heat injuryby treatment of cream containing 1% compound B.

FIG. 21 shows the wound healing on STZ diabetic rats after heat injuryby treatment of cream containing 2% compound B.

FIG. 22 shows the wound healing on STZ diabetic rats after heat injuryby treatment of cream containing 4% compound B.

FIG. 23 shows the wound healing on STZ diabetic rats after heat injuryby treatment of cream containing 1% compound B, but evaluated visually.

FIG. 24 shows the wound healing on STZ diabetic rats after heat injuryby treatment of cream containing 2% compound B, but evaluated visually.

FIG. 25 shows the wound healing on STZ diabetic rats after heat injuryby treatment of cream containing 4% compound B, but evaluated visually.

FIG. 26 shows the comparison photographs (treated and control) made inthe above tests by digital epiluminescence microscopic technique.

FIG. 27 shows the hsp72 level of the samples obtained in the previoustests determined by Western blotting at the treatments with creamscontaining 1, 2 and 4% compound B.

FIG. 28 shows the hsp72 levels determined by immunohistochemicalanalysis (treated and control) on SCID mice exposed to UV-B ray andtreated with compound B.

FIG. 29 shows the hsp72 levels determined by Western blotting on SCIDmice skin biopsy samples exposed to UV-B ray and treated with compoundB.

DETAILED DESCRIPTION OF THE INVENTION

The entire disclosure of U.S. patent application Ser. No. 08/860,582,filed Dec. 7, 1999, is expressly incorporated by reference herein.

Hydroxylamine Derivatives of the Invention

Hydroxylamine derivatives, the tautomeric forms of which are representedby formulae (I) and (II), can be used in accordance with the inventiondescribed herein. In the above formulae A is an alkyl, substitutedalkyl, aralkyl, aralkyl substituted in the aryl and/or in the alkylmoiety, aryl, substituted aryl, heteroaryl or substituted heteroarylgroup,

Z is a covalent bond, oxygen or ═NR³ wherein R³ is selected from thegroup consisting of hydrogen, an alkyl, substituted alkyl, aryl,substituted aryl, aralkyl and aralkyl substituted in the aryl and/or inthe alkyl moiety,

R is an alkyl or substituted alkyl,

X in the tautomer of formula (I) is halogen or a substituted hydroxy oramino, monosubstituted amino or disubstituted amino group and X in thetautomer of formula (II) is oxygen, imino or substituted imino group and

R′ is hydrogen, an alkyl, substituted alkyl, aryl, substituted aryl,aralkyl, aralkyl having substituted aryl or alkyl moiety, acyl orsubstituted acyl group,

and the compounds of formula (I) optionally contain intramolecular ringstructures formed by coupling X and a reactive substituent.

Where alkyl” is mentioned, it means straight or branched alkyl groupscomprising short and long chains as well.

The typical number of carbon atoms of a preferred short chain alkylgroup ranges from 1 to 8 and might be methyl-, ethyl-, propyl-,isopropyl-, butyl-, isobutyl-, sec-butyl-, pentyl-, tert-pentyl-;hexyl-, heptyl-, and octyl- groups and the like, more preferably 1 to 6and might be methyl-, ethyl-, propyl-, isopropyl-, butyl-, isobutyl-,sec-butyl-, pentyl-, tert-pentyl-; and hexyl- groups.

The typical number of carbon atoms of a preferred long chain alkyl groupranges from 9 to 21 and might be nonyl-, decyl-, undecyl-, dodecyl-,tridecyl, tetradecyl-, pentadecyl-, hexadecyl-, heptadecyl, octadecyl-,nonadecyl-, eicosyl- and heneicosyl- groups and the like, morepreferably from 9 to 17 and might be nonyl-, decyl-, undecyl-, dodecyl-,tridecyl, tetradecyl-, pentadecyl-, hexadecyl-, and heptadecyl- groups.

A preferred cycloalkyl group means a cycloalkyl group having a shortcycloalkyl chain ranges from 3 to 8 and might be cyclopropyl-,cyclobutyl-, cyclopentyl-, cyclohexyl-, cycloheptyl- and cyclooctyl-groups and the like, more preferably from 3 to 7 and might becyclopropyl-, cyclobutyl-, cyclopentyl-, cyclohexyl- and cycloheptyl-groups.

Optionally substituted aryl or alkyl means an aryl- or alkyl grouphaving one or more substituents such as cyano-, hydroxyl-, short chainalkyl- (e.g. methyl-, ethyl-, propyl-, isopropyl-, butyl-, isobutyl-,sec-butyl-, pentyl-, tert-pentyl-, hexyl-, heptyl-, octyl- and thelike), short chain alkoxy- (e.g. methoxy-, ethoxy-, propoxy-,isopropoxy-, butoxy-, isobutoxy, sec-butoxy-, tert-butoxy-, pentyloxy-,tert-pentyl-oxy-, hexyloxy- and the like), aryl- (e.g. phenyl-,naphthyl-, and the like), nitro-, amino-, mono-(short chainalkyl)-substituted amino- (e.g. methyl, ethyl, propyl, isopropyl,tert-butyl)-amino and the like, di-(short chain alkyl)-substitutedamino- (e.g. dimethylamino-, diethylamino-, dipropylamino-,diisopropylamino-, dibutylamino-, dipentylamino-, dihexylamino- and thelike), monohalogen-, dihalogen- or trihalogen (short chain)-alkyl- (e.g.chloromethyl, 2,2-dichloroethyl, trifluoromethyl- and the like) group orhalogen atom (e.g. fluoro-, chloro-, bromo-, and iodine atom) and thelike as well.

A preferred aralkyl group means a short chain alkyl group as writtenabove, substituted with one or more (optionally substituted) aryl groupsand might be benzyl-, benzhydryl-, trityl-, 1-phenyl-ethyl-,2-phenylethyl-, 2-benzhydryl-ethyl-, 3-phenylpropyl-,1-methyl-2-phenyl-ethyl-, 1-phenylbutyl-, 4-tritylbutyl-,1,1-dimethyl-2-phenylethyl-, 4-phenylbutyl-, 5-phenylpentyl-,6-phenylhexyl- groups and the like, more preferably lower alkyl groupfrom 1 to 4 carbon atom, substituted with a phenyl group and might bebenzyl-, 1-phenylethyl-, 2-phenylethyl-, and 1-methyl-2-phenylethylgroups. A preferred aryl group might be phenyl-, naphthyl-, pentalenyl-,anthracenyl- groups and the like, more preferably phenyl- and naphthylgroups.

A preferred 3-8 membered, more preferably 5-8 membered, N-containingsaturated heterocyclic group means a saturated heterocyclic groupcontaining 1-4 nitrogen atoms and might be aziridiriyl-, azetidinyl-,oxaziranyl-, pyrrolidinyl-, imidazolidinyl-, pyrazolidinyl-,perhydro-thiazolyl-, perhydro-isoxazolyl-, piperidinyl-, piperazinyl-,perhydro-pyrimidinyl-, perhydro-pyridazinyl-, morpholinyl-,perhidro-1H-azepinyl- groups and the like.

A preferred heteroaryl group means an unsaturated, 3-8 membered, morepreferably 5-6 membered, 1-4 N-containing unsaturated hetero-monocyclicgroup and might be pyrrolyl-, pyrrolinyl-, imidazolyl-, pyrazolyl-,pyridyl- group and its N-oxide, pirimidinyl-, pyrazinyl-, pyridazinyl-,triazolyl-, tetrazolyl-, dihydrotriazinyl- group and the like; or meansunsaturated, 1-5 N-containing condensed heterocyclic group and might beindolyl-, isoindolyl-, indolizinyl-, benzimidazolyl-, quinolyl-,isoquinolyl-, indazolyl-, benzotriazolyl-, tetrazolopyridyl-,tetrazolopyri-dazinyl-, dihydro-triazolopyridazinyl- group and the like;or means a 3-6 membered, more preferably 5-6 membered, 1-2 oxygen- and1-3 N-containing unsaturated hetero-monocyclic group and might beoxazolyl-, isoxazolyl-, oxadiazolyl- (e.g. 1,2,4-oxadiazolyl- andothers) group and the like; or means unsaturated, 1-2 oxygen- and 1-3N-containing condensed heterocyclic group and might be benzoxazolyl-,benzoxadiazolyl- group and the like; or means a 3-8 membered, morepreferably 5-6 membered, 1-2 sulfur- and 1-3 N-containing unsaturatedhetero-monocyclic group and might be thiazolyl-, 1,2-thiazolyl-,thiazolinyl-, thiadiazolyl- group and the like; or means a 3-8 membered,more preferably 5-6 membered, one S-containing unsaturatedhetero-monocyclic group and might be thienyl- group; or means oneO-containing unsaturated heteromonocyclic group and might be furyl-group; or means unsaturated, 1-2 sulfur- and 1-3 N-containing condensedheterocyclic group and might be benzothiazolyl-, benzothiadiazolyl-group and the like.

A preferred “acyl” group when taken in itself or forming part of anacylated group, preferably means an acyl group which might be a shortchain alkanoyl- (e.g. formyl-, acetyl-, propionyl, butyryl- and thelike), a short chain alkoxy-carbonyl- (e.g. methoxy-carbonyl-,ethoxy-carbonyl-, propoxy-carbonyl-, butoxy-carbonyl-,tert-butoxy-carbonyl- and the like), a short chain alkyl-sulphonyl-(e.g. methyl-sulphonyl-, ethyl-sulphonyl- and the like), aryl-sulphonyl-(e.g. phenyl-sulphonyl- and the like), aroyl- (e.g. benzoyl, naphthoyl-and the like), aryl-(short chain alkanoyl)- (e.g. phenyl-acetyl-,phenyl-propionyl- and the like), cyclo-(short chain alkyl)-(short chainalkanoyl)- (e.g. cyclohexyl-acetyl and the like), aryl-(short chainalkoxy)-carbonyl- (e.g. benzyloxy-carbonyl and the like),aryl-carbamoyl- (e.g. phenyl-carbamoyl-, naphthyl carbamoyl- and thelike), cycloalkyl-carbamoyl- (e.g. cyclohexyl-carbamoyl- and the like),hetero-monocyclic sulphonyl- (e.g. thienyl-sulphonyl-, furyl-sulphonyl-and the like) group; and the acyl group can be optionally substitutedwith 1-3 substituents as written above in the “optionally substituted”section.

A preferred T-amino-alkyl group means a short chain alkyl groupcontaining substituted N-atom in the T-position of the alkyl chain andin which the alkyl chain is optionally substituted with one or moresubstituents, preferably with one or two halogen (e.g. chloro-, bromo-,fluoro-, iodo-), hydroxyl group or acylated hydroxyl group, where theacyl group has been defined earlier; more preferably with one or twoshort chain alkyl groups and the “alkyl” definition is the same aswritten above. The N-atom in the T position of the alkyl chain can besubstituted with one or two short chain alkyl substituents, preferablymethyl-, ethyl-, tert-butyl- and the like; with cycloalkyl carbamoyl-(e.g. cyclohexyl-carbamoyl- and the like), more preferably the N-atomcan be a part of a saturated heterocyclic group which contains 1-4nitrogen atoms and might be aziridinyl-, azetidinyl-, oxaziranyl-,pyrrolidinyl-, imidazolidinyl-, pyrazolidinyl-, perhydro-thiazolyl-,perhydro-izoxazolyl-, piperidinyl-, piperazinyl-, perhydro-pyrimidinyl-,perhydro-pyridazinyl-, morpholinyl-, perhidro-1H-azepinyl- groups andthe like; the N-atom in the T position can be substituted with an arylgroup (e.g. phenyl and the like), and can be quaternized by a shortchain alkyl substituent or oxidized as well.

If desired, the free bases of the general formulae (I) and (II) may betransformed to acid addition salts by reacting with organic acids andmay be acetate, maleate and the like; or by reacting with inorganicacids and may be hydrochloride, hydrobromide, hydroiodide, sulphate,phosphate and the like; or by reacting with amino acids and may bearginine-salt, glutamic acid salt and the like.

In a non-limiting embodiment of the hydroxylamine derivative ofstructure (I), Z is a covalent bond and X is a halogen, preferablychloro or bromo. Preferred compounds belonging to this group has a A (i)aralkyl or aralkyl having substituted aryl moiety, preferably phenylalkyl or phenyl alkyl having one or more substituents, preferablyalkoxy; (ii) aryl or substituted aryl, preferably phenyl or substitutedphenyl, preferably substituted phenyl containing one or more of alkyl,halogen, haloalkyl, alkoxy or nitro group; (iii) naphthyl; (iv) anN-containing heteroaryl group, including those which may be condensedwith a benzene ring, preferably piridyl; (v) an S-containing heteroarylgroup or (vi) an O-containing heteroaryl group. Preferred compoundsbelonging to this group has as R (i) T-amino-alkyl, (ii) T-amino-alkylhaving mono or disubstituted amino moiety; (iii) T-amino alkyl havingsubstituted alkyl moiety; (iv) T-amino alkyl having mono ordisubstituted amino moiety and also substituted alkyl moiety, with ahydroxy or acyloxy group being preferred substituent group for the alkylmoeity. Of the T-amino-alkyl group of (i) to (iv), particularlypreferred are those with 3-8 carbon atom alkyl moiety.

Certain types of the hydroxylamine derivative of structure (I) havingcovalent bond as Z and halogen as X are disclosed in the U.S. Pat. Nos.5,147,879, 5,328,906 and 5,296,606. These compounds can be prepared byprocedures described in the cited US patents, preferably bydiazotization of the corresponding X═NH₂ derivatives in the presence ofthe appropriate hydrohalide. The starting compounds can be obtained byknown procedures described e.g. in Hungarian Patent No. 177,578 (1976),namely by coupling an amidoxime of structure 1 (R¹═R²═H) with e.g. areactive derivative of structure 2 in the presence of a base, and can bediazotized usually without isolation or purification. The terminalgroups A and R of the compounds can be further amidified or derivatized,as desired.

In another non-limiting embodiment of the hydroxylamine derivative ofstructure (I), Z is covalent bond and X is a substituted hydroxy groupOQ, wherein Q is an unsubstituted or substituted alkyl or aralkyl group.In a preferred embodiment, Q is a linear or branched alkyl. In thesecompounds, A is aryl or heteroaryl, preferably a N-containingheteroaromatic group; and R is preferably a (i) T-amino-alkyl, (ii)T-amino-alkyl having mono or disubstituted amino moiety; (iii) T-aminoalkyl having substituted alkyl moiety; (iv) T-amino alkyl having mono ordisubstituted amino moiety and also substituted alkyl moiety, with ahydroxy or acyloxy group being preferred substituent group for the alkylmoiety. Of the T-amino-alkyl of (i) to (iv), particularly preferred arethose with 3-8 carbon atom alkyl moiety.

A special group of the hydroxylamine derivatives of structure (I),wherein Z is covalent bond and X is OQ, is of structure (I′). Structure(I′) contains a ring closed through the hydroxy group. These compoundsrepresent a cyclic form of the compounds of structure (I), wherein R isa —CH₂—CH(OH)—R″,R″ being a liner or branched alkyl, or a substitutedlinear or branched alkyl, preferably T-amino-alkyl which is optionallysubstituted on its amino group and preferably contains C₁₋₅ straight orbranched alkyl chain. Most preferably, R″ is an T-amino-alkyl mono- ordisubstituted on the amino group, wherein the amino-substituents,independently from each other may be one or two straight or branchedalkyl or cycloalkyl, or the two amino-substituents, together with theadjacent N-atom form a 3 to 7, preferably 5 to 7-membered hetero ring,which optionally contains additional hetero atom. Of these, preferredcompounds have A that is a phenyl, substituted phenyl, N-containingheteroaryl, substituted N-containing heteroaryl, S-containingheteroaryl, or substituted S-containing heteroaryl.

Hydroxylamine derivatives of structure (I) having covalent bond as Z andOQ as X have been disclosed in the Hungarian Patent Application No.2385/1992. These compounds can be prepared from the correspondinghalogen derivatives of the above group (hydroxylamine derivatives ofstructure (I), wherein Z is covalent bond and X is halogen) byprocedures described in the Hung. Pat. Appln. No. 2385/1992, e.g., byreaction with alkoxides, or by alkaline ring closure for the cycliccompounds of structure (I′).

In a non-limiting embodiment of the hydroxylamine derivative ofstructure (I), Z is covalent bond and X is NR¹R², wherein R¹ and R²,independently from each other, are H, a linear or branched alkyl, asubstituted linear of branched alkyl, cycloalkyl, or R¹ and R², togetherwith the nitrogen atom attached thereto, form a saturated ringcontaining 3 to 7 members, preferably 5-7 membered saturated ring.

Of the compounds described in the immediately preceding paragraph,especially preferred are those wherein R is a (i) T-amino-alkyl, (ii)T-amino-alkyl having mono or disubstituted amino moiety; (iii) T-aminoalkyl having substituted alkyl moiety; (iv) T-amino alkyl having mono ordisubstituted amino moiety and also substituted alkyl moiety, with ahydroxy or acyloxy group being preferred substituent group for the alkylmoiety. Of the T-amino-alkyl group of (i) to (iv), particularlypreferred are those with 3-8 carbon atom alkyl moiety. Of thesecompounds, further preferred ones have A that is (i) aralkyl or aralkylhaving substituted aryl moiety, preferably phenyl alkyl or phenyl alkylhaving one or more substituents, preferably alkoxy; (ii) aryl orsubstituted aryl, preferably phenyl or substituted phenyl, preferablyphenyl containing one or more of alkyl, halogen, haloalkyl, alkoxy,nitro, or acylamino group; (iii) naphthyl; (iv) an N-containingheteroaryl group, including those which may be condensed with a benzenering, preferably piridyl; (v) an S-containing heteroaryl group or (vi)an O-containing heteroaryl group.

Hydroxylamine derivatives of structure (I) having covalent bond as Z andNR¹R² as X include both known and new derivatives. Compounds where X isNH₂ are disclosed in Hungarian Patent No. 177578 (1976) and can besynthesized by alkylation of unsubstituted amidoxime derivatives ofstructure 1 (structure 1, wherein R¹—R²═H) with a reactive derivative ofstructure 2 in presence of a base.

A special group of the hydroxylamine derivatives of structure (I),wherein Z is covalent bond and X is NR¹R², is provided by structure(I″). Structure (1″) represents a cyclic form of structure (I) whichcontains a ring closed through NR¹R² group. These compounds can bederived from compounds of structure (I), wherein R² is H and R isCH₂—CH(OH)—R″, R″ being a linear or branched alkyl or a substitutedlinear or branched alkyl.

Of the compounds of structure (1″), preferred are those wherein A is (i)aryl or substituted aryl, preferably phenyl or substituted phenyl,preferably substituted phenyl containing one or more of alkyl, halogen,haloalkyl, alkoxy, amino or nitro group; (ii) naphthyl; (iii) anN-containing heteroaryl group, including those which may be condensedwith a benzene ring; (iv) S-containing heteroraryl group; and (v)O-containing heteroaryl group. Especially preferred of these compoundscontain R″ which is (i) T-amino-alkyl having mono or disubstituted aminomoiety, or (ii) T-amino alkyl having mono or disubstituted amino moietyand also substituted alkyl moiety, preferably the alkyl moiety ofT-amino-alkyl of (i) and (ii) contains 1-5 carbon atoms. Especiallypreferred are the T-amino-alkyl group having disubstituted amino moiety,wherein the substitutents, together with the nitrogen atom attachedthereto, form a 3-7 member, preferably 5-7 member, saturatedheterocyclic ring. The heterocyclic ring may contain additionalheteroatom(s). In these T-amino-alkyl groups the amino-substituent ispreferably a linear or branched alkyl group or cycloalkyl. In thecompounds of the general formula (I″) R¹ is hydrogen, unsubstituted orsubstituted straight or branched alkyl, cycloalkyl, unsubstitutedaralkyl or aralkyl substituted in the aryl- and/or alkyl moiety.

The compounds of structure (I″) can be prepared by the ring closurebetween atoms N(4)-C(5). The required open chain derivatives arecompounds of structure (I) wherein Z is a covalent bond, X is ═NR¹R²,wherein R¹ is as defined in connection with the compounds of the formula(I″) above, R² is H and R is a group of the formula —CH₂—CHY⁵—R″ whereinY⁵ represents a leaving group, e.g., a halogen atom. Such derivativescould be obtained from the corresponding Y⁵═OH compounds with inorganichalogenating agents, e.g., thionyl chloride or phosphorus pentachloride.The halogenation can be carried out with or without an inert solvente.g. benzene, chloroform, tetrahydrofurane etc., usually by boiling.After removing the excess of the reagent, e.g., by evaporation of thethionyl chloride, the crude halogen derivative is cyclized—either afteror without isolation or purification—by treatment with a strong base,e.g., potassium butoxide in t-butanol to give compound I″, which isfinally isolated and purified by standard procedures (extraction,recrystallization, etc.).

In a non-limiting embodiment of the hydroxylamine derivative ofstructure (I), Z is oxygen and X is OQ, wherein Q is an alkyl,substituted alkyl, aralkyl, or aralkyl having substituted aryl orsubstituted alkyl moiety. The alkyl or substituted alkyl that is Q haspreferably 1-4 carbon atoms. Of these compounds, preferred ones have Athat is an alkyl or substituted alkyl, preferably 1-4 carbon atoms, oraralkyl or aralkyl having substituted aryl or substituted alkyl moiety.Of these compounds, preferred have R that is (i) T-amino-alkyl, (ii)T-amino-alkyl having mono or disubstituted amino moiety; (iii) T-aminoalkyl having substituted alkyl moiety; (iv) T-amino alkyl having mono ordisubstituted amino moiety and also substituted alkyl moiety, with ahydroxy or acyloxy group being preferred substituent group for the alkylmoiety.

These hydroxylamine derivatives of structure (I), wherein Z is oxygenand X is OQ, can be obtained in the reaction of O-substitutedhydroxylamines having structure 6 (see e.g., Ger. Off. 2,651,083 (1976))and orthoesters having structure 7. The condensation is usually carriedout in the regent itself, as a solvent, preferably by boiling. Afterevaporation, the product is isolated by crystallization, occasionally,(if there is an amine function in the side chain R) in the form of acidaddition salt.

In a non-limiting embodiment of the hydroxylamine derivative ofstructure (I), Z is oxygen and X is NR¹R², wherein R¹ and R²,independently from each other, are H, a linear or branched alkyl, asubstituted linear or branched alkyl, cycloalkyl, aryl, substitutedaryl, or R¹ and R², together with the nitrogen atom attached thereto,form a saturated ring containing 3 to 7 members, preferably 5-7 memberedsaturated ring. Of these compounds especially preferred are thosewherein R is a (i) T-amino-alkyl, (ii) T-amino-alkyl having mono ordisubstituted amino moiety; (iii) T-amino alkyl having substituted alkylmoiety; (iv) T-amino alkyl having mono or disubstituted amino moiety andalso substituted alkyl moiety, with a hydroxy or acyloxy group beingpreferred substituent group for the alkyl moiety. Of the T-amino-alkylgroup of (i) to (iv), particularly preferred are those with 3-8 carbonatom alkyl moiety. Of these compounds, it is preferred that A is (i)alkyl or substituted alkyl; (iii) aralkyl or aralkyl having substitutedaryl and/or substituted alkyl moiety; or (iv) aryl or substituted aryl,preferably phenyl or substituted phenyl.

Preparation of the compounds can be prepared as described herebelow,wherein the methods depend on the nature of X, namely whether X is anunsubstantiated amino (NH₂) or a substituted amino functionality.

Preparation of the compounds where X is NH₂ can be accomplished by theaddition of hydroxylamine of structure 6 to an organic cyanate ofstructure A-O—CN (see, e.g., Chem. Ber. 98, 144 (1965)). The reaction iscarried out preferably in an inert organic solvent, usually at roomtemperature. The isolation often requires chromatographic purification.

The compounds having X that is monosubstituted amino group (e.g., NHR¹)are prepared from known haloformimidates of structure 9 (sec e.g.Houben-Weil, “Methoden der Organischen Chemie”, Band E/4, p. 544 (1983)and a compound of structure 6, in the presence of an organic base (e.g.,triethylamine) or an inorganic base, such as sodium carbonate in aninert solvent, as benzene, tetrahydrofurane, etc., followed by standardwork-up and purification procedures.

Derivatives where X is a disubstituted amino group are prepared by thereaction of a secondary amine of structure 5 with a compound ofstructure 1, where Z is oxygen and X is OQ (preparation of thesederivatives is described above). These amination reactions are performedin polar organic solvents, e.g., ethanol, by refluxing, if necessary.

In another non-limiting embodiment of the hydroxylamine derivative ofstructure (I), Z is ═NR³, wherein R³ is hydrogen, an alkyl, substitutedalkyl, aryl, substituted aryl, aralkyl, or aralkyl having substitutedaryl or substituted alkyl moiety; and X is NR¹R², wherein R¹ and R²,independently from each other, are H, a linear or branched alkyl, asubstituted linear of branched alkyl, aryl or substituted aryl,cycloalkyl, or R¹ and R², together with the nitrogen atom attachedthereto, form a saturated ring containing 3 to 7 members, preferably 5-7membered saturated ring.

Of these compounds, it is further preferred that A is an alkyl,substituted alkyl, aralkyl, aralkyl, aralkyl having substituted aryl orsubstituted alkyl moiety, aryl, or substituted aryl group. Preferred Rfor compounds belonging to this group of hydroxylamine derivative is (i)T-amino-alkyl, (ii) T-amino-alkyl having mono or disubstituted aminomoiety; (iii) T-amino alkyl having substituted alkyl moiety; (iv)T-amino alkyl having mono or disubstituted amino moiety and alsosubstituted alkyl moiety, with a hydroxy or acyloxy group beingpreferred substituent group for the alkyl moiety. It is preferred thatthe alkyl moiety of T-amino-alkyl of (i) to (iv) contain 3-8 carbonatoms.

The hydroxylamine derivatives of structure (I), wherein Z is NR³ and Xis NR¹R², can be prepared by aminolysis of the corresponding isoureaderivatives belonging to a group of compounds described above (thisgroup corresponds to the hydroxylamine derivatives of structure (I)having Z is oxygen and X is NR¹R²) with ammonia or a primary orsecondary amine. The reaction is carried out preferably in a polarsolvent, e.g., water or ethanol, using excess of the amine.Alternatively, haloformamides of structure 10 (Houben-Weil “Methoden derOrganischen Chemie,” Band E/4, page 553 (1983)) can be reacted with acompound having structure 6 in the presence of an organic or inorganicbase to give compounds of this group as well. The reaction carried outin inert organic solvent, usually at ambient temperature.

The compounds wherein R is a group of the formula (b) wherein R⁷ isacyl, are prepared by esterifying the corresponding compounds containinghydrogen as R⁷. The alkyl- or aryl esters are usually obtained by usingan acid chloride or anhydride in the presence of a tertiary amine or aninorganic base, preferably in an inert solvent.

Another group of hydroxylamine derivatives useful in the presentinvention have structure (ID, which represents the tautomeric form ofthe compounds of structure (I). In a non-limiting embodiment of thehydroxylamine derivative of structure (II), Z is covalent bond and X isoxygen. Preferred compounds belonging to this group has A that is (i)alkyl, aralkyl or aralkyl having substituted aryl or alkyl moiety; (ii)aryl or substituted aryl, preferably phenyl or substituted phenyl havingone or more substituents, preferred substituent groups including analkyl, haloalkyl or alkoxy group; (iii) an N-containing heteroarylgroup, preferably piridyl; or (iv) S-containing heteroaryl group. Forcompounds belonging to this group, preferred R is (i) T-amino-alkyl,(ii) T-amino-alkyl having mono or disubstituted amino moiety; (iii)T-amino alkyl having substituted alkyl moiety; (iv) T-amino alkyl havingmono or disubstituted amino moiety and also substituted alkyl moiety,with a hydroxy or acyloxy group being preferred substituent group forthe alkyl moiety. Of the T-amino-alkyl group of (i) to (iv),particularly preferred are those with 3-8 carbon atom alkyl moiety.Preferred compounds of this group has R′ that is hydrogen, an alkyl,substituted alkyl, aryl, substituted aryl, aralkyl, or aralkyl havingsubstituted aryl or alkyl moiety.

Compounds belonging to this group are disclosed in the Hungarian PatentApplication No. 2385/1992. The routes for their preparation aredescribed therein, most preferably, they can be obtained by acylation ofO-substituted hydroxylamine derivatives having structure 6 (see also,e.g., Ger. Off. 2,651,083 (1976)) with an acid chloride having structure11. This route can be employed also for the preparation of these newderivatives, where R′ is other than hydrogen, using compound ofstructure 12—instead of structure 6—as starting material.

In another non-limiting embodiment of the hydroxylamine derivative ofstructure (II), Z is chemical bond; X is ═NR⁴, wherein R⁴ is H, analkyl, substituted alkyl, aryl, substituted aryl, aralkyl, aralkylhaving substituted aryl or substituted alkyl group, cycloalkyl; and R′that is an alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, oraralkyl having substituted aryl or substituted alkyl moiety. Among thesecompounds preferred are those wherein (A) is (i) aralkyl or aralkylhaving substituted aryl moiety, preferably phenyl alkyl or phenyl alkylhaving one or more substituents, preferably alkoxy; (ii) aryl orsubstituted aryl, preferably phenyl or substituted phenyl, preferablysubstituted phenyl containing one or more alkyl, haloalkyl or nitrogroup; (iii) naphthyl; (iv) an N-containing heteroaryl group, preferablypiridyl; or (v) S-containing heteroaryl group. Preferred compoundsbelonging to this group as R (i) T-amino-alkyl, (ii) T-amino-alkylhaving mono or disubstituted amino moiety; (iii) T-amino alkyl havingsubstituted alkyl moiety; (iv) T-amino alkyl having mono ordisubstituted amino moiety and also substituted alkyl moiety, with ahydroxy or acyloxy group being preferred substituent group for the alkylmoiety. of the T-amino-alkyl group of (i) to (iv), particularlypreferred are those with 3-8 carbon atom alkyl moiety.

These compounds can be prepared either by O-alkylation of aN,N′-disubstituted amidoxime of structure 13 with a chemical compoundhaving structure 2 (for the reaction conditions, see preparation ofcompounds of structure I, wherein Z is covalent bond and X is NR¹R²), orby O-acylating an N,O-disubstituted hydroxylamine of the formula 12 withan imidoyl halide of the formula 16, the reaction being carried out inan inert solvent, preferably in the presence of an organic or inorganicacid scavanger.

The compounds wherein R is a group of the formula (b) wherein R⁷ isacyl, are prepared by esterifying the corresponding compounds containinghydrogen as R⁷. The alkyl- or aryl esters are usually obtained by usingan acid chloride or anhydride in the presence of a tertiary amine or aninorganic base, preferably in an inert solvent.

In another non-limiting embodiment of the hydroxylamine derivative ofstructure (II), Z is oxygen and X is oxygen. Preferred compoundsbelonging to this group has A that is an alkyl, substituted alkyl,aralkyl, or aralkyl with substituted aryl or alkyl moiety. R ispreferred to be (i) T-amino-alkyl, (ii) T-amino-alkyl having mono ordisubstituted amino moiety; (iii) T-amino alkyl having substituted alkylmoiety; (iv) T-amino alkyl having mono or disubstituted amino moiety andalso substituted alkyl moiety, with a hydroxy or acyloxy group beingpreferred substituent group for the alkyl moiety. Of the T-amino-alkylgroup of (i) to (iv), particularly preferred are those with 3-8 carbonatom alkyl moiety. Preferred compounds of this group has R′ that ishydrogen, an alkyl, substituted alkyl, aryl, substituted aryl, aralkyl,or aralkyl with substituted aryl or alkyl moiety.

Compounds belonging to this group are disclosed in Hung. PatentApplication No. 1756/95 (filed Jun. 15, 1995). They are prepared byacylation of a hydroxylamine having structure 6 or structure 12 with achloroformate having structure 14, in a similar manner as with thesimple acid chlorides, as described for the synthesis of compounds ofstructure II wherein Z is covalent bond and X is oxygen. The reactionrequires the presence of a base, inorganic or organic, and can beperformed in an inert solvent, e.g., in chloroform. The side-productsalt is removed, e.g., by extraction with water, and the product isisolated from the organic solution.

In yet another non-limiting embodiment of the hydroxylamine derivativeof structure (II), Z is oxygen; X is ═NR⁴, wherein R⁴ is alkyl,substituted alkyl, aralkyl, aralkyl having substituted aryl orsubstituted alkyl group, aryl, substituted aryl, heteroaryl orsubstituted heteroaryl group. In these compounds A is preferably alkyl,substituted alkyl, aryl, substituted aryl, most preferably unsubstitutedor substituted phenyl, aralkyl or aralkyl with substituted aryl or alkylmoiety, and R is preferably T-aminoalkyl, which suitably contains ahydroxy or acyloxy group in the alkyl chain, and is optionallysubstituted on the amine nitrogen, wherein the alkyl chain of the saidT-aminoalkyl group preferably contains 3 to 8 carbon atoms. In thesecompounds R′ is preferably alkyl, aryl or aralkyl which groups may beunsubstituted or substituted.

These compounds are N-substituted analogues of hydroxylamine derivativesof structure (I), wherein Z is oxygen and X is NR¹R², and can beprepared, similarly from haloformimidates having structure 9 and achemical compound having structure 12, in the presence of an organicbase (e.g., triethylamine) or inorganic base. e.g. sodium carbonate inan inert solvent, as benzene, tetrahydrofurane etc., followed bystandard work-up and purification procedures.

In another non-limiting embodiment of the hydroxylamine derivative ofstructure (II), Z is ═NR³, wherein R³ is selected from the groupconsisting of hydrogen, an alkyl, substituted alkyl, aryl, substitutedaryl, aralkyl, and aralkyl having substituted aryl or substituted alkylmoiety; and X is oxygen. Preferred compounds of this group have A thatis (i) aralkyl or aralkyl having substituted alkyl or aryl moiety,preferably phenylalkyl or phenylalkyl having one or more substituents;(ii) aryl or substituted aryl, preferably phenyl or substituted phenyl,preferably substituted phenyl containing one or more of alkyl, alkoxy,halogen, haloalkyl or nitro group; (iii) an N-containing heteroarylgroup; or (iv) an alkyl or substituted alkyl, linear or branched,preferably containing 4 to 12 carbon atoms, or (v) a cycloalkyl group.Preferred compounds belonging to this group has (i) T-amino-alkyl, (ii)T-amino-alkyl having mono or disubstituted amino-moiety; (iii) T-aminoalkyl having substituted alkyl moiety; (iv) T-amino alkyl having mono ordisubstituted amino moiety and also substituted alkyl moiety, with ahydroxy or acyloxy group being preferred substituent group for the alkylmoiety. Of the T-amino-alkyl group of (i) to (iv), particularlypreferred are those with 3-8 carbon atom alkyl moiety. In thesecompounds R′ is preferably hydrogen, an alkyl, substituted alkyl,aralkyl or aralkyl having substituted aryl or alkyl moiety, aryl,substituted aryl, acyl or substituted acyl group.

These compounds are disclosed in a co-pending Hungarian PatentApplication No. 1756/95 and can be prepared by reaction of ahydroxylamine compound having structure 6 or structure 12 with anisocyanate having structure 15, in an inert solvent, usually by simplestirring of the mixture at room temperature for 2-24 hours. Finally, theproducts are isolated—after evaporation of the solvent—preferably bycrystallization.

In a non-limiting embodiment of the hydroxylamine derivatives ofstructure (II), Z is ═NR³, wherein R³ is selected from the groupconsisting of hydrogen, an alkyl, substituted alkyl, aryl, substitutedaryl, aralkyl, and aralkyl having substituted aryl or substituted alkylmoiety; X is ═NR⁴, wherein R⁴ is H, an alkyl, substituted alkyl, aryl,substituted aryl, aralkyl, aralkyl having substituted aryl orsubstituted alkyl group, cycloalkyl,; and R′ is an alkyl, substitutedalkyl, aralkyl, or aralkyl having substituted aryl or substituted alkylmoiety, or aryl or substituted aryl. Preferred compounds belonging tothis group have as R³ hydrogen, alkyl or substituted alkyl R⁴ ishydrogen or an aryl group, A is alkyl, substituted alkyl, aryl orsubstituted aryl, or aralkyl, which may be substituted in the aryland/or alkyl moiety. Of these compounds, preferred ones have R that is(i) T-amino-alkyl, (ii) T-amino-alkyl having mono or disubstituted aminomoiety; (iii) T-amino alkyl having substituted alkyl moiety; (iv)T-amino alkyl having mono or disubstituted amino moiety and alsosubstituted alkyl moiety, with a hydroxy or acyloxy group beingpreferred substituent group for the alkyl moiety. Of the T-amino-alkylgroup of (i) to (iv), particularly preferred are those with 3-8 carbonatom alkyl moiety.

Preparation of compounds belonging to this group can be accomplished byaminolysis of the of the corresponding isourea derivatives (compoundshaving structure (H), wherein Z is oxygen and X is NR⁴) with a primaryor secondary amine or ammonia. The reaction is carried out preferably ina polar solvent, e.g., water or ethanol, using an excess of the amine.Alternatively, haloformamidines having structure 10 can react with acompound of structure 12 in the presence of an organic or inorganic basein inert solvents, usually at their boiling point.

One non-limiting embodiment of the hydroxylamine derivative of structure(I) defines a novel group of compounds, wherein X is halogen, preferablya chloro or bromo; Z is a chemical bond and A is a group of the formula(a) wherein Y¹ is halo, alkoxy, a nitro group or a haloalkyl group,preferably haloalkyl containing 1-4 carbon atoms; and n is 1, 2, or 3;or O-containing heteroaryl, preferably furyl, S-containing heteroaryl(preferably thienyl), or N-containing heteroaryl group (preferablypiridyl, quinolyl, or isoquinolyl) which may be condensed with a benzenering and R is a group having structure (b), wherein R⁵ and R⁶,independently from each other, are H, a linear or branched alkyl,preferably a substituted linear or branched alkyl, preferably C₁₋₄alkyl, or cycloalkyl, or R⁵ and R⁶, when taken together with thenitrogen atom attached thereto, form a 3 to 7, preferably 5 to 7,membered saturated heterocyclic ring, Y⁶ is —OR⁷, wherein R⁷ is H or anacyl group, preferably alkyl carbonyl, substituted alkyl carbonyl, arylcarbonyl or substituted aryl carbonyl, or aminoacyl or substitutedaminoacyl; k is 1, 2 or 3; and m is 1, 2, or 3, with the proviso, thatwhen A is piridyl or naphtyl, or a group of the formula (a) wherein Y¹is halo or alkoxy, then R⁷ is other than H. These compounds mayoptionally contain as A an N-containing heteroaromatic group withN-quaternary C₁₋₄ alkyl or the oxide of the said N-containingheteroaromatic group and/or an R wherein the ring formed by the terminalgroups R⁶ and R⁷ is an N-quaternary or N-oxidized saturated heterocyclicring. Preferred are among these compounds those wherein A is a group ofthe formula (a) wherein Y¹ is trifluoromethyl. This group of thehydroxylamine derivatives of the formula (I) also includes the opticallyactive stereoisomers of the compounds wherein X is halo, A is piridyl, Zis a chemical bond, and R is the group of the formula (b) wherein R⁵ andR⁶ independently from each other are H, straight or branched alkyl,preferably C₁₋₄ alkyl or cycloalkyl, or R⁵ and R⁶, together with theadjacent N atom form a 3 to 7 membered, preferably 5 to 7 memberedheterocyclic ring, Y⁶ is —OR⁷, wherein R⁷ is aminoacyl, k is 1, 2 or 3and m is 1, 2 or 3.

These novel compounds can be prepared using procedures that areanalogous to those described in U.S. Pat. Nos. 5,147,879; 5,328,906; and5,296,606. For example:

Derivatives where both R⁵ and R⁶ are other than hydrogen, are preparedby the diazotization of the corresponding NH₂ derivatives (thehydroxylamine derivatives of structure (I), wherein Z is covalent bondand X is NH₂) in the presence of the appropriate hydrogen halide,similarly to the procedure described in U.S. Pat. Nos. 5,147,879;5,328,906; and 5,296,606. The starting compounds can be obtained also bya known procedure described, e.g., in Hungarian Patent No. 177578,namely by coupling an amidoxime having structure 1, wherein R¹ and R² ofstructure 1 is H, with e.g. a reactive derivative having structure 2 inthe presence of a base, and can be diazotized usually without isolationor purification.

If in the desired structure R⁷ is H and m is 1, the synthesis can beaccomplished by the reaction of an oxyrane having structure 3 and aminehaving structure 4. This procedure also can be used for the synthesis ofR⁵═H derivatives.

Compounds where R is a group of the structure (b), and where R⁷ in thisgroup is an acyl group, are prepared by the esterification of thecorresponding R⁷═H derivatives. Alkyl or aryl esters are usuallyobtained with an acid chloride or anhydride in the presence of atertiary amine or an inorganic base, preferably in an inert solvent, orin certain cases by the Schotten-Bauman procedure using aqueousinorganic base in a two-phase system. For the preparation of theaminoacyl esters, carboxyl-activated N-protected amino acid derivatives(e.g., active esters) are used as reagents in procedures basically knownfrom the peptide chemistry. This coupling also requires the presence ofa base (e.g. triethylamine). The isolation and purification of theproducts are performed by using standard preparative techniques; thefinal preparation is often in the form of a salt with appropriateinorganic or organic acids. Starting from chiral amino acids, theproducts are frequently diastereomers, possessing the second chiralcenter in the R group. During the isolation, these diastereomers oftenseparate, and the product can be obtained in stereo-pure form.

Compounds having structure (I) wherein Z is chemical bond, X is halo,preferably chloro or bromo A is a group of the formula (c) and R is agroup of the formula (d); one or both of Y² and Y³, from which at leastone must be present in the molecule, are oxygen, or an alkyl orsubstituted alkyl having 1-4 carbon atoms; k is 1, 2, or 3; and m is 1,2, or 3. Y² and Y³ are attached by the dotted line, which means theoptional presence of these substituents, are also novel hydroxylaminederivatives. When the compound is a mono- or bivalent cation, the anionthereof is one or two halide, preferably iodide ion.

These hydroxylamine derivatives are prepared by the chemicalmodifications (i.e., N-oxidation or quaternerization) of the terminalpyridine and/or piperidine groups in their unsubstituted precursors. Forthe oxidation, preferably peracids, e.g. substituted perbenzoic acidsare used in inert solvents (e.g., chloroform, dichloromethane). If bothoxidizable groups are present in the molecule, mono- or dioxides mayform depending on the quantity of the reagent used. At the end of thereaction, the excess reagent is decomposed and the product is isolatedby evaporation. The quaternerization can be accomplished with alkylhalides (e.g., methyliodide), preferably by refluxing the reagent in asuitable solvent, e.g., acetone. The product is often insoluble in themedium, and can be isolated by simple filtration.

Yet another novel group of compounds belonging to the hydroxylaminederivatives having structure (I) are those wherein Z is a chemical bond.A is aralkyl, substituted aralkyl, preferably phenylalkyl which may haveone or more alkoxy, preferably alkoxy having 1 to 4 carbon atom, phenyl,substituted phenyl having one or more substituents, preferredsubstituent groups including an alkyl, preferably alkyl or haloalkylhaving 1 to 4 carbon atom, halo, acylamino or nitro group; or aN-containing heteroaryl group, which may be condensed with benzene ring,preferably pyrrolyl, pyridyl, isoquinolyl or quinolyl, or a sulfurcontaining heteroaromatic group, preferably thienyl, wherein theheteroaryl groups may be substituted with one or more alkyl, preferablyalkyl having 1 to 4 carbon atoms; X is —NR¹R², wherein R¹ and R²,independently from each other, are H, a linear or branched alkyl, asubstituted linear or branched alkyl, preferably alkyl having 1 to 6carbon atoms, a cycloalkyl or R¹ and R² taken together with the nitrogenatom attached thereto may form a 3 to 7, preferably 5 to 7, memberedsaturated hetero ring; R is a group of the formula (e), wherein R⁵ andR⁶, independently from each other, are H, a linear or branched alkyl, ora substituted linear or branched alkyl, preferably alkyl having 1 to 4carbon atoms, or cycloalkyl, or R⁵ and R⁶ taken together with thenitrogen atom attached thereto form a 3-7, preferably 5-7 memberedsaturated hetero ring, which may contain additional hetero atoms andsubstituents, the substituents being preferably alkyl having 1 to 4carbon atoms; Y⁴ is H or an alkyl or substituted alkyl having 1-4 carbonatoms; Y⁵ is H, or an alkyl or substituted alkyl having 1-4 carbonatoms, or OR⁷ wherein R⁷ is H or an acyl; k is 1, 2, or 3; and m is 1,2, or 3, with the proviso that

when A is phenyl which is unsubstituted or substituted with halogen oralkoxy; or phenylalkyl substituted with alkoxy; or a pyridyl group, andR⁷ is H, then at least one of R¹ and R² is other than H, or

when A is phenyl which is unsubstituted or substituted with halogen oralkoxy; phenylalkyl substituted with alkoxy; or pyridyl, and R¹ and R²are each H, then R⁷ is other than H.

The compounds wherein X is an NH₂ derivative, are prepared—similarly tothe above-mentioned procedure—by the reaction of the correspondingintermediates having structure 1, wherein R¹ and R² of structure 1 areH, with a compound having structure 2. The alkylating agent (havingstructure 2) may contain hydroxyl and/or amino substituents. Thereaction requires the presence of an inorganic or organic base, in apreferable manner alcoholic alcoholate solution is used as medium andbase. The products are often isolated in the form of salt with asuitable organic or inorganic acid.

Another group of the above novel compounds is characterized by R¹ andR², one or both of them being other than H in these derivatives. Suchstructures can be prepared in two ways:

(i) An amidoxime having structure 1, which already contains the requiredsubstituents R¹ and/or R², can react with a reactive compound ofstructure 2, similarly to the procedure described in the previousparagraph. The substituted amidoximes of structure 1, used as startingmaterials, are known from the literature [Chem. Rev. 62, 155-183(1962)].

(ii) Substitution of the halogen atoms in the compounds having structure(I), wherein Z is covalent bond and X is halogen, by an amine ofstructure 5 can result in similar compounds as well. In the case ofderivatives bearing an OH substituent in the R group (Y⁴═OH), thishydroxyl group has to be protected before, and deprotected after thesubstitution reaction, otherwise formation of the cyclic derivatives ofstructure (I′) is favored. For the protection, acetyl type protectinggroups, e.g., tetrahydropyranyl group, have proven most satisfactory.The protection is carried out by the reaction of the unprotectedcompound with dihydropyrane, followed by the halogen/amine displacement,which usually requires refluxing in a solvent, e.g., in alcohol. Thedeprotection of the product, finally, can be accomplished b acidictreatment, e.g., by boiling the ethanolic solution in the presence ofe.g. p-toluenesulphonic acid.

As mentioned, a group of the novel compounds also includes those whereinY⁵ is an acyloxy group. They can be prepared by acylation of thecorresponding Y⁵═OH derivatives, which are either known from theliterature (e.g., Hung, Patent No. 177578) or described in the presentinvention. The reactions can be accomplished identically to what isdescribed for the analogous halo derivatives, wherein R⁷ is an acylgroup (method (iii)).

The novel hydroxylamine derivatives of the formula (I) also includethose wherein Z is oxygen or an ═NR³ group wherein R³ is anunsubstituted or substituted alkyl group, X is —NR¹R². R¹ and R²independently from each other are hydrogen, unsubstituted or substitutedstraight or branched alkyl, unsubstituted or substituted aryl,preferably phenyl or unsubstituted or substituted aralkyl group or R¹and R² when taken together with the nitrogen atom attached thereto, forma 3 to 7 membered, preferably 5 to 7 membered saturated heterocyclicring which optionally contains one or more hetero atoms. In thesecompounds A is an unsubstituted or substituted alkyl or unsubstituted orsubstituted aryl, preferably phenyl or substituted phenyl group or anunsubstituted or substituted aralkyl group and R is a group of theformula (b) wherein R⁵ and R⁶, independently from each other are H,straight or branched alkyl, preferably C₁₋₄ alkyl, or cycloalkyl, or R⁵and R⁶, together with the N-atom attached thereto, form a 3 to7-membered, preferably 5 to 7-membered saturated heterocyclic ring, Y⁶is H or —OR⁷, wherein R⁷ is H or acyl, preferably unsubstituted orsubstituted alkylcarbonyl or arylcarbonyl, k is 1, 2, or 3 and m is 1, 2or 3.

The novel hydroxylamine derivatives, wherein Z is oxygen and X is —OR,wherein Q is an unsubstituted or substituted alkyl or unsubstituted orsubstituted aralkyl group, A is an unsubstituted or substituted alkoxygroup or an unsubstituted or substituted aralkyl group and R is a groupof the formula (b), wherein R⁵ and R⁶, independently from each other areH, straight or branched alkyl, preferably C₁₋₄ alkyl, or cycloalkyl, orR⁵ and R⁶, together with the N-atom attached thereto, form a 3 to7-membered, preferably 5 to 7-membered saturated heterocyclic ring, Y⁶is H or —OR⁷, wherein R⁷ is H or acyl, preferably unsubstituted orsubstituted alkylcarbonyl or arylcarbonyl, k is 1, 2 or 3 and m is 1, 2or 3, fall also within the scope of compounds of formula (I).

R⁵ and R⁶, independently from each other, are H, straight or branchedalkyl, preferably C₁ alkyl or cycloalkyl, or R⁵ and R⁶, when takentogether with the N atom attached thereto form a 3 to 7-membered,preferably 5 to 7-membered heterocyclic ring, Y⁶, is H or —OR⁷, R⁷ is Hor acyl, preferably unsubstituted or substituted alkylcarbonyl orarylcarbonyl, k is 1, 2 or 3 and m is 1, 2 or 3.

Another group of the novel hydroxylamine derivatives of the formula (I)is represented by those wherein A is unsubstituted or substituted aryl,preferably phenyl or N-containing heteroaromatic group, preferablypiridyl or S-containing heteroaromatic group, Z is a chemical bond. X is—OQ wherein Q is C₁₋₄ alkyl and R is a group of the formula (b), whereinR⁵ and R⁶, independently from each other are H, straight or branchedalkyl preferably C₁₋₄ alkyl or cycloalkyl or R⁵ and R⁶, when takentogether with the N atom attached thereto form a 3 to 7-membered,preferably 5 to 7-membered heterocyclic ring. Y⁶ is H, k is 1, 2 or 3and m is 1, 2 or 3.

These compounds are prepared by the reaction of the correspondinghydroxylamine derivatives of the formula (I) wherein X is halo and thecorresponding alcoholates, preferably in an alcohol corresponding to thealcoholate, preferably by refluxing. The reaction mixture is treatedwith methods known in the art and the product is isolated bychromatography or salt-forming.

The novel hydroxylamine derivatives of the formula (II) also include thegroup of compounds wherein X is oxygen, A is C₁₋₂₀ straight or branchedalkyl, unsubstituted or substituted aryl, preferably phenyl orhalophenyl, unsubstituted or substituted aralkyl, naphtyl orN-containing heteroaromatic group, preferably piridyl, Z is a chemicalbond. R′ is H, C₁₋₄ alkyl or aralkyl, preferably phenylalkyl, R is agroup of the formula (b), wherein R⁵ and R⁶, independently from eachother, are H, straight or branched alkyl, preferably C₁₋₄ alkyl orcycloalkyl, or R⁵ and R⁶, when taken together with the N atom attachedthereto form a 3 to 7-membered, preferably 5 to 7-membered heterocyclicring, Y⁶ is H or —OR⁷, R⁷ is H, k is 1, 2 or 3 and m is 1, 2 or 3, withthe proviso, that when A is other than alkyl and R′ is H, Y⁶ is H.

The novel compounds wherein Z is a covalent bond, oxygen or an ═NR³group wherein R³ is hydrogen or an unsubstituted or substituted alkylgroup, X is ═NR⁴, wherein R⁴ is hydrogen, an unsubstituted orsubstituted alkyl or unsubstituted or substituted aryl, preferablyphenyl group, or substituted or unsubstituted aralkyl, preferablyphenylalkyl, fall also within the scope of compounds or formula (II). Inthese compounds A is an unsubstituted or substituted alkyl or anunsubstituted or substituted aryl preferably phenyl or substitutedphenyl, or unsubstituted or substituted aralkyl, preferably phenylalkyl,or cycloalkyl, R′ is an unsubstituted or substituted alkyl orunsubstituted or substituted aryl, preferably phenyl, or unsubstitutedor substituted aralkyl, preferably phenylalkyl, R is a group of theformula (b), wherein R⁵ and R⁶, independently from each other, are H,straight or branched alkyl, preferably C₁₋₄ alkyl or cycloalkyl, or R⁵and R⁶, when taken together with the N atom attached thereto form a 3 to7-membered, preferably 5 to 7-membered heterocyclic ring, Y⁶ is H or—OR⁷, R⁷ is H or acyl, preferably unsubstituted or substitutedalkylcarbonyl or arylcarbonyl, k is 1, 2 or 3 and m is 1, 2 or 3.

Novel hydroxylamine derivatives are also those of the formula (II)wherein X is oxygen, A is unsubstituted or substituted alkyl,unsubstituted or substituted aralkyl, preferably phenylalkyl, Z isoxygen, R′ is alkyl or aralkyl, preferably phenylalkyl, R is a group ofthe formula (b), wherein R⁵ and R⁶, independently from each other, areH, straight or branched alkyl, preferably C₁₋₄ alkyl or cycloalkyl, orR⁵ and R⁶, when taken together with the N atom attached thereto form a 3to 7-membered, preferably 5 to 7-membered heterocyclic ring, Y⁶ is H or—OR⁷, R⁷ is H or acyl, preferably unsubstituted or substitutedalkylcarbonyl or arylcarbonyl, k is 1, 2 or 3 and m is 1, 2 or 3.

The hydroxylamine compounds of the formula (II), wherein X is oxygen andZ is ═NH, are also novel compounds.

One group of these compounds is formed by those wherein A isunsubstituted or substituted alkyl, cycloalkyl, unsubstituted orsubstituted aralkyl, preferably phenylalkyl, unsubstituted phenyl orphenyl substituted with halo, alkyl, haloalkyl, alkoxy or nitro, R is agroup of the formula (b), wherein R⁵ and R⁶, independently from eachother, are H, straight or branched alkyl, preferably C₁₋₄, alkyl orcycloalkyl, or R⁵ and R⁶, when taken together with the N atom attachedthereto form a 3 to 7-membered, preferably 5 to 7-membered heterocyclicring, Y⁶ is H or —OH, k is 1, 2 or 3 and m is 1, 2 or 3.

Another group of these compounds is formed by those wherein A is a groupof the formula (a), wherein Y¹ is haloalkyl, preferably trifluoromethyland n is 1, 2 or 3, R′ is H and R is a group of the formula (b), whereinR⁵ and R⁶, independently from each other, are H, straight or branchedalkyl, preferably C₁₋₄ alkyl or cycloalkyl, or R⁵ and R⁶, when takentogether with the N atom attached thereto form a 3 to 7-membered,preferably 5 to 7-membered heterocyclic ring, Y⁶ is H or —OH, k is 1, 2or 3 and m is 1, 2 or 3.

The novel hydroxylamine derivatives according to the invention alsoinclude the cyclic compounds of the formula (I″), wherein A isunsubstituted phenyl or phenyl substituted with halo or nitro, orN-containing heteroaryl, R¹ is H and R″ is an T-amino-alkyl group mono-or disubstituted on the amino group, the alkyl chain of which having 1to 5 carbon atoms and the amino substituents, independently from eachother, may be one or two straight or branched alkyl or cycloalkyl, orthe two amino-substituents, together with the N atom adjacent thereto,form a 3 to 7-membered, preferably 5 to 7-membered saturatedheterocyclic ring, or a C₁₋₄ alkyl N-quaternary derivative thereof, withthe proviso, that when A is 3-piridyl, R″ is different from1-piperidinylmethyl.

Method of Increasing Molecular Chaperon Expression in Cells

The present invention relates to a method of enhancing expression of amolecular chaperons in cells, by treating cells and tissues with aneffective amount of a hydroxylamine derivative. Hydroxylaminederivatives, the tautomeric forms of which are represented by formulae(I) or (II), can be used for this method. Structures of the formulae (I)and (II) are discussed in details in the preceding section entitled“Hydroxylamine Derivatives of the Invention”.

In one non-limiting embodiment of the invention, a method of increasingmolecular chaperon expression in cells exposed to stress is provided. Inthis method, the cells exposed to a physiological stress which inducesexpression of molecular chaperons by the cells are treated with aneffective amount of hydroxylamine derivatives, the tautomeric forms ofwhich are represented by formulae (I) and (II), after occurrence of thestress. The hydroxylamine derivatives increase the expression ofmolecular chaperons in these cells beyond the amount induced by thephysiological stress.

In another non-limiting embodiment of the invention, the cells aretreated with the hydroxylamine derivatives before they are exposed to aphysiological stress. The hydroxylamine derivatives increase themolecular chaperon expression beyond the amount induced by thephysiological stress.

The term “molecular chaperon,” as used herein, refers to protein whichassists other proteins to fold into correct or active conformations,usually by non-covalently binding to the proteins. Chaperons not onlyassist in the correction and restoration of proteins newly synthesizedbut also of those that have been denatured or misfolded. Molecularchaperons include, among others, heat shock proteins (hsp) from whichhsp70 and hsp72 are of especial importance in connection with theinvention, as well as the IgG heavy chain binding protein (BiP), andglucose regulated proteins (grp). Examples of hsp and grp include, butare not limited to, those belonging to the following classes: hsp70,hsp60, hsp90, grp94, grp80, and hsp27.

Preferably, eukaryotic cells which are treated with the hydroxylaminederivatives are mammalian cells, more preferably human cells. The term“eukaryotic cell” refers to both eukaryotic cells that are in vitro(cells that are outside a living organism, e.g. in culture condition)and in vivo (cells that are within a living organism, e.g., cellscomprising tissues and organs).

From the eucaryotic cells of a living organism, neurons, muscle cells,vessel wall cells, especially endothelial cells, epithelial cells andcells of the immune system can preferably be treated in accordance withthe method of the invention. Plant cells, including cells of livingplant organisms can also preferably be treated in accordance with themethod of the invention.

Under the term “physiological stress,” as used herein, conditions orfactors affecting the cell which would induce the “stress response” ofthe cell, e.g., induction of chaperon protein synthesis should beunderstood. Physiological stresses include factors that cause injury tocells or those disturbing homeostatic balance of cells. These are, forexample, the metabolic, oxidative, local mechanical stresses or stressescaused by hypoxia, ischemia, heat shock, radiation or toxic materials.An important form of metabolic stress is caused by diabetes mellitus.

Another important appearance form of physiological stresses includesthose leading to the formation of free radicals or increase of thequantity of cytokines in the environment of cells. Injuries in cellsthat are associated with various pathological conditions provideexamples of physiological stresses.

In one non-limiting embodiment of the invention, the physiologicalstress that induces cells to express molecular chaperon as a response tophysiological stresses leading to cardiovascular, vascular, cerebral,allergic, immune, autoimmune diseases, viral and bacterial infections,skin and mucosal diseases or diseases of renal tubuli of epithelialorigin or causing conditions to be treated by cosmetical interventions.

Such cardiovascular diseases include most preferably atherosclerosisprovoked by physiological stress, coronarial diseases, or cardiovasculardiseases caused by hypertonia or pulmonary hypertonia.

Characteristic cerebral diseases are, among others, those caused by thecerebrovascular ischemia provoked by physiological stress, stroke,traumatic head injury, senile neurodegenerative diseases, especiallysenile dementia, AIDS dementia, alcohol dementia, Alzheimer's disease,Parkinson disease or epilepsy.

Characteristic diseases provoked by skin and mucosal diseases are thedermatological diseases or ulcerous diseases of the gastrointestinalsystem.

During the above diseases, the physiological stress induces chaperonexpression in the cells, however, this effect is not sufficient enoughto protect against cell damages caused by the diseases. The treatmentwith the above hydroxylamine derivatives which is associated withenhancement of chaperon expression or increase of chaperon activitymakes possible the elimination of structural deviations caused by thedisease and thus, regeneration of cells.

In one non-limiting example of the invention, the physiological stressis heat shock or exposure of cells to unusually high temperature. Inanother non-limiting example of the invention, the physiological stressis cellular injury associated with ischemia. Ischemic lesion of cells,especially heart muscle and cerebral cells is caused by cardiovasculardisorders caused by vascular occlusion or rapture, such as coronary orcerebral thrombosis or vascular occlusion, stroke, embolism, or chronicvascular spasm. Ischemia induces “stress response” in cells, resultingin increased amount of hsp, which in turn protect the cells againstdeleterious effects of ischemia. (Mestril, R. et al, J. Mol. Cell.Cardiol, 27: 45 (1995).

By treating these cells with an effective amount of hydroxylaminederivatives to those cells, molecular chaperon expression in the cellscan be increased beyond the amount induced by ischemic condition as wellas the activity of molecular chaperons can be enhanced.

In connection with this matter, when an organ is taken out of an animalfor transplantation, such removal is a physiological stress causinginjury to the cells comprising that organ, inducing chaperon expression.In such a case, administration of the hydroxylamino derivatives beforeor after the organ removal could increase the amount of chaperonproduced by the cells of the organ or the activity thereof thusproviding cytoprotective effect.

Neuronal injuries, besides ischemia, can be induced by many otherstresses as well, which induce molecular chaperon production in theneuronal cells. In addition, excitotoxic neuronal injuries also induceproduction of molecular chaperons by neuronal cells and are includedwithin the term “physiological stress.”

In yet another example of the invention, physiological stress isprovided by the toxic mediators of inflammation, such as oxidativeradicals and cytokines, such as TNF, which are produced by macrophages.Cells exposed to increased amount of these toxic mediators are shown toexpress an increased amount of hsp, which in turn provide protection tothese cells against the toxicity. (Kantengwa, S. et al, Semin. Immun. 3:49-56 (1991). Various inflammatory diseases including pulmonaryinflammatory conditions, such as adult distress syndrome, induceexpression of hsp by the cells, which in turn exert cytoprotectiveeffect. (Jacquier-Salin, M. R. et al., Experientia 50: 1031-1038(1994)). When the amount of molecular chaperons in cells is increasedbeyond that induced by TNF and reactive oxygen species, the cells can bebetter protected against these cytotoxic factors and better enable torepair the damages caused by these.

Factors affecting the physiological state of cell membranes, includingcell membrane fluidity, also provide examples of physiological stress.Increase of molecular chaperon expression in these cells beyond thatinduced by disturbance of the physiological state of cell membranes canprovide better protection and also allow the cells to repair the cellmembranes.

The phrase “an effective amount of the hydroxylamine derivatives,” asused herein in connection with enhancing molecular chaperon productionin cells under physiological stress, or cells which will be subsequentlyexposed to physiological stress, refers to an amount which will increasethe expression of molecular chaperon beyond the level induced by thephysiological stress alone. Such amount can be readily determined by oneskilled in the art. Preferably, for cells in vitro, the effective amountis between 10⁻⁶-10⁻³ M. More preferably, the effective amount is between10⁻⁶-5×10⁻⁴ M.

When administering to an animal, the effective amount varies dependingon various factors, such as a mode of administration, but determiningeffective range is within the skill of one skilled in the art and willnot require undue experimentation. For example, when the hydroxylaminederivative is administered intravenously, the effective amount ispreferably between 0.1-10 mg/kgbw, more preferably 0.5-2.0 mg/kgbw; andwhen administered orally, the effective amount is preferably between10-500 mg/kgbw, more preferably between 50-100 mg/kgbw.

An increase in the molecular chaperon expression in cells can bedetected using well established laboratory procedures such as Northernor Western blotting procedure.

An example of the Western blotting technique that can be used is setforth herein: Cells are cultured in vitro at 37° C. in Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% fetal calf serum(GIBCO) in 5% CO₂. Hydroxylamine derivatives the tautomeric forms ofwhich are represented by formulae (I) and (II) can be added to the cellculture, for example, 10⁻⁵ M of the compound is administered to cells 16hours before the physiological stress, or after the time periodfollowing the physiological stress. However, the concentration of thehydroxy-lamine derivative, as well as the time of the administration ofthat compound, can be varied as desired by the experimental design.

Six hours after the heat shock, cells are washed two times inphosphate-buffered saline solution (PBS) then scrapped from the surfaceof the culture dishes in PBS. Then cells are spun for 5 min. at 1500 rpmand taken up in 100 μl modified solubilizing buffer (Molecular Cloning,A Laboratory Manual, Ed. Sambrook, Fritsche, Maniatis, Bold SpringHarbor Laboratory Press (1989)) containing 50 mM Tris-HCl, pH8.0; 5 mMEDTA; 150 mM NaCl; 15 Tritox N-100; 1 PMSF; 2:g/ml aprotinin; 1 μg/mlchymostatin; 1 μg/ml pepstatin; and sonicated for 3×2o sec (2 min.intervals, setting 8).

Protein concentration is then determined from 5 μl samples by theBradford assay (M. M. Bradford, Anal. Biochem., 72: 248-254 (1976)) inthree parallel. Samples are adjusted to 100 μg/30 μl proteinconcentration with the above buffer and the next buffer so that thefinal concentration of the components in the buffer in the sample willbe: 110 mM Tris-HCl pH 6.8, 8.3 mM mercaptoethanol, 3% SDS, 3% glyceroland some bromophenol blue and shaken at room temperature for 30 min. Thesample thus obtained can then be used for to run a gel-electrophoresis.

When chaperon enhancing effect of the hydroxylamine derivatives of theinvention is examined for cells in vivo, a physiological stress isapplied to an animal, e.g., ischemia or STZ-induced diabetes. In case ofischemia, myocardial ischemia can be induced in an animal as describedin Example 8; and diabetic condition can be induced as described inExample 10. A hydroxy-lamine derivative of the present invention can beadministered to the animal before it is exposed to physiological stress,during the stress or afterwards. As stated previously, the timing of theadministration can be varied according to an experimental design.

The following steps of protein preparation from relevant tissuesobtained from the animals thus treated are carried out at 0-4 C.Tissues, such as liver tissues (about 15-20 g) are homogenized with adomestic mixer for 2 min. in 80 ml lysis buffer solution containing 50mM Tris-HCl pH 8.0, 5 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100and 1-1 mM protease inhibitors (PMSF, benzamidine, amino-caproic-acid).The homogenate is then centrifuged at 20000×g for 30 min. in a SorvallRC 28S centrifuge.

Protein concentration of the preparation is determined by the Bradfordassay and adjusted to 5 mg/ml. The samples containing 1.8 mg protein aresolubilized before gel-electrophoresis with 0.6 ml buffer containing 110mM Tris-HCl pH 6.8, 8.3 mM mercaptoethanol, 3% SDS, 3% glycerol and somebromophenol blue and shaken at room temperature for 30 min.

The protein samples obtained from cell cultures or from animal tissues,both of which are described above, are used for electrophoresis andsubsequent immunoblotting (both procedures are well known in the art anddescribed in detail in Molecular Cloning, A Laboratory Manual, Ed.Sambrook, Fritsche, Maniatis, Bold Spring Harbor Laboratory Press(1989); Protein Blotting Protocols for the Immobilon-P TransferMembrane, 3. Laboratory Manual, Millipore; and U. K. Laemmli, Nature:227: 680-685 (1970).

For example, electrophoresis can be carried out according to Laemmli (U.K. Laemmli, Nature, 227:680-685 (1970)) on 8-18% polyacrylamide gel atconstant voltage 50 V for overnight. Proteins are either stained withCoomassie Brilliant Blue R-250 or transferred to Immobilone PVDF(Millipore) at constant current (300 mA) for 3 hours at 4° C. intransfer buffer (10 mM CAPS, pH 11, 10% methanol) (Protein BlottingProtocols for the Immobilon-P Transfer Membrane, 3. Laboratory Manual,Millipore). After transfer, non-specific sites of the membrane areblocked with 2% bovine serum albumin (BSA) in TPBS (phosphate bufferedsaline with 0.1% Tween 20) for overnight at 4° C. The blot can then beincubated with an antibody directed as a molecular chaperon, e.g. GRP94monoclonal antibody (SPA-850, StressGen) diluted 1:3000, with HSP60monoclonal antibody (SPA-600, StressGen) with 1:2700 dilution, withHSP72 monoclonal antibody (C92F34-5, StressGen) diluted 1:1250 or withHSP90 monoclonal antibody (AC88, StressGen) diluted 1:2000, for 1 hourat room temperature. Then the membrane is washed with TPBS buffer forone hour, and incubated with horseradish peroxidase conjugated anti-rat(Sigma, 1:4000 dilution, for grp-94) or anti-mouse (Sigma, adsorbed withhuman and rat serum proteins, 1:3000 dilution, for Hsp60, HSP72 orHSP90) secondary antibody for additional 1 hour respectively. Aftersuccessive washing with TPBS the membrane is developed with ECL(enhanced chemiluminescence) system (Amersham).

The changes in the stress protein content can be quantified using aBio-Rad densitometer (Model 1650) and a Hewlett-Packard Integrator (HP3394A). Dilution series are prepared from protein solution containingknown amount of chaperon, the above process is repeated with thedilutions and the chaperon concentration of the test samples aredetermined from the calibration curve obtained from the dilution tests.

Northern hybridization is another experimental procedure available fordetermining the level of molecular chaperon enhancement (by measuringthe mRNA level) by the hydroxylamine derivatives of the invention. Thecells or tissues can be obtained as described in connection with theWestern blotting procedure. Total RNA from those cells and tissues canbe extracted using RNAgents (Promega) according to the manufacturer'sinstructions (Protocols and Applications Guide, 2nd edition, 1991,Promega Corporation). The frozen tissue samples (about 50 to 100 mgeach) are homogenized in 1.0 denaturing 4M guanidine-thiocyanate; 42 mMsodium citrate: 0.83 m β-mercaptoethanol; 0.1% Nonidet P-40) at 4° C.(Brinkman-homogenization). Then 1/10 vol. 3M sodium acetate (pH 4.0) isadded and the homogenate are extracted with acidic phenol(phenol:chloroform:isoamylalcohol 25:24:1) for 10 seconds by vortex. Thesample is incubated on ice for 15 minutes, and then centrifuged (4 C; 20min., 10,000 ×g). The aqueous phase is then transferred to a newEppendorf-tube the process is repeated and the aqueous phase isprecipitated at −20° C. overnight with equal volume of isopropanol.Following centrifugation (4 C; 20 min. 10,000×g) the precipitate iswashed twice with 95% ethanol and dried at room temperature. The RNA issuspended in 20 μl diethyl-pyrocarbonate (DEPC)-treated water and theconcentrate is measured at 260-280 nm by spectrophotometry. Eight μg oftotal RNA is run on formaldehydeagarose gel by capillary transfer, theRNA on the gel is blotted onto nylon membrane according to themanufacture's instructions (Zeta-Probe. GT, BioRad).

In individual samples, the molecular chaperon mRNA content is comparedwith the mRNA level of the glyceraldehyde-3-phosphate dehydrogenase(GAPDH) gene in the samples. cDNA probes (for example, full length humanhsp70 cDNA when the mRNA probed is hsp70, and Apa-NcoI fragment of therat GAPDH cDNA) are labeled with alpha ³²P CTP using Random Prime DNALabeling Kit (USB). Radiolabeled DNA fragments are purified on SephadexG-50 (Pharmacia) column as described (Ausubel et al. (eds)): CurrentProtocols in Molecular Biology: JOHN WILEY & SONS: 1987).

Prehybridizations are carried out at 65° C. in H-buffer (0.25M Na₂HPO₄,pH 7.2, 7% SDS) for 15 minutes. Hybridizations are carried out overnight(65 C; H-buffer) with isotope labeled cDNA probe concentration of atleast 10⁶ cpm/ml. The membrane is then washed with 20 mM Na₂HPO₄, pH7.2, 5% SDS (65 C; 2×15 min.) and evaluated by autoradiography. The samemembrane is used for probing the hsp70 mRNA and the GAPDH and mRNAmeasurement used as internal standard.

The present invention further includes a method of treating orpreventing various pathological conditions, i.e. diseases associatedwith the functioning of chaperon system and damages in the membranes ofcells and cell-organelli by administering an effective amount ofhydroxylamine derivatives, the tautomeric forms of which are representedby structures (I) and (II) to control pathological conditions in theorganism. In the pathological conditions, characteristic molecularchaperon expression is induced in the cells. Increased molecularchaperon expression in those cells can assist them in repairing thedamages caused by the pathological conditions and also in restoring thecellular homeostatic balance.

Such pathological conditions include ischemia, tumorous diseases,infections caused by pathogenic microorganisms, autoimmune diseases anddermatosis.

As used herein, “treating” refers to an amelioration in the clinicalcondition of the subject, and does not necessarily indicate that acomplete cure is achieved. An amelioration refers to a decreasedduration of illness or severity of illness, or subjective improvement inthe quality of life of the subject or a prolonged survival of thepatient.

An effective amount of hydroxylamine of the invention for treatmentrefers to an amount sufficient to result in the amelioration of clinicalcondition as described above. An effective amount depends on factorssuch as the route of administration and can easily be determined by oneskilled in the art. The hydroxylamine derivatives of the presentinvention can be administered parenterally or orally, preferably orallyor topically, and the effective amount is 10-500 mg/kgbw. Morepreferably, the effective amount is 20-100 mg/kgbw.

By using the method of treatment according to the present invention, themyocardium, brain tissues and kidney can be protected against tissuedamage or necrosis caused by ischemia, wherein the method comprisesadministering to a subject an effective amount of hydroxylaminederivatives of the invention to decrease, prevent, or reverse thedeleterious effect of prolonged ischemia.

The present invention includes use of a hydroxylamine derivatives, thetautomeric forms of which are represented by formulae (I) and (II) tomanufacture a medicament for the treatment of pathological conditionsdescribed herein.

In a method for measuring the protective effect of the hydroxylaminederivatives animal test is used as set forth herein. Rats areanaesthetized with sodium-pentobarbital (Nembutal 60 mg/kg body weight,i.p.) and artificially ventilated with room air (2 ml/100 g; 54stroke/minutes) via tracheotomy. The right carotid artery is thencatheterized and connected to a pressure transducer (BPR-01, Stoelting)for the measurement of systemic arterial blood pressure (BP) by means ofa preamplifier (Hg-02, Experimetria). Hydroxylamine derivatives of theinvention are administered via cannule to jugular vein (i.v.) or orally(p.o.). Heart rate (HR) is measured by a cardiotachometer (HR-01,Experimetria); and the electrocardiogram (ECG standard lead II) isrecorded on a devices recorder (MR-12, Medicor) by means of subcutaneoussteel needle electrodes. The chest is opened by a left thoracotomy andthe heart is then exteriorized by a gentle pressure on the right side ofthe rib cage. A compression was applied under the main left coronaryartery as described by Selye et al. (1960). The heart is carefullyreplaced in the chest and the animal left to recover. Rectal temperatureis monitored and kept constant at 37 C. The experimental protocol isinitiated with a 15 minute stabilization period. If sustained bloodpressure less than 70 mmHg or arrhythmia were observed during thisperiod the animal was excluded from further experimentation. Myocardialischemia is then induced with coronary occlusion for 5 minutes andreperfusion is allowed for 10 minutes.

During the entire experiment, blood pressure (BP), heart frequency (HR)and EKG are continuously registered on a multiscriptor (R61-6CH,Medicor*). Hydroxylamine derivatives are administered at 5 to 60 minutesbefore the occlusion by i.v. or p.o. treatment. The doses of thehydroxylamine derivative can be 0.5; 0.75; 1.0 mg/kg i.v. and 100 mg/kgof body weight p.o., while the reference substance Bepridil is given ina dose of 1.0 mg/kg i.v. The mean duration of ventricular tachycardia(VT) and/or ventricular fibrillation (V F) during the first 3 minutes ofreperfusion is measured and analyzed.

The present invention also includes a method of maintaining a cellmembrane fluidity, when the cell membrane fluidity is affected as aresult of a physiological stress. The method comprises the treatment ofa cell or cell-organellum having altered membrane fluidity with aneffective amount of hydroxylamine derivatives to restore the fluidity ofsaid membrane. The experimental protocol set forth in connection withExample 9 (Steady State DPH fluorescence anisotropy) can be used fordetermining the effect of a hydroxylamine derivative of the invention onthe cell membrane fluidity.

As mentioned, the present invention includes a method of treatingpathological conditions associated with cell membrane or cell-organellummembrane. One example of such pathological condition is provided bydiabetes mellitus as well as the diseases associated with mitocondriumdamage, such as ALS (amyotrophic lateral sclerosis), Alzheimer disease,Parkinson disease, Huntington disease (HD), certain cardiomyopathies,such as those of toxic origin, caused by alcohol or heavy metals,inflammatory or viral cardiomyopathy or autoimmune cardiomyopathy.Hydroxylamine derivatives, the tautomeric forms of which are representedby structures (I) and (II), can be used in this method.

The method of the present invention can be used in the treatment oftumorous diseases, the method comprising administering an effectiveamount of hydroxylamine derivatives to the tumorous organism to preventformation or growth of tumors. Hydroxylamine derivatives, the tautomericforms of which are represented by structures (I) and (II), can be usedin this method.

Pharmaceutical and Cosmetical Compositions Containing the HydroxylamineDerivatives

As already mentioned, the invention also relates to the use ofhydroxylamine derivatives of the general formulae (I) and (II),including the optically active strereoisomers thereof, in thepreparation of pharmaceutical compositions (and optionally cosmeticalcompositions) useful in the treatment of cardiovascular, vascular,allergic, immune, autoimmune diseases, diseases caused by viral orbacterial infection, tumorous, skin and mucous diseases and renal tubulediseases provoked by physiological stress as well as those conditionscaused also by physiological stresses which can be treated by cosmeticalintervention, wherein formulae (I) and (II), or its salts, including theoptically active stereoisomers thereof.

A is an alkyl, substituted alkyl, aralkyl, aralkyl substituted in thearyl and/or in the alkyl moiety, aryl, substituted aryl, heteroaryl orsubstituted heteroaryl group,

Z is a covalent bond, oxygen or ═NR³ wherein R³ is selected from thegroup consisting of hydrogen, an alkyl, substituted alkyl, aryl,substituted aryl, aralkyl, or aralkyl substituted in the aryl and/or inthe alkyl moiety,

R is an alkyl or substituted alkyl,

X in the tautomer of formula (I) is halogen or a substituted hydroxy oramino, monosubstituted amino or disubstituted amino group and

X in the tautomer of formula (II) is oxygen, imino or substituted iminogroup and

R′ is hydrogen, an alkyl, substituted alkyl, aryl, substituted aryl,aralkyl, aralkyl having substituted aryl and/or alkyl moiety, acyl orsubstituted acyl group,

and the compounds of formula (I) optionally contain intramolecular ringstructures formed by coupling X and a reactive substituent.

By using these compounds, compositions for both preventive and curativepurposes can be prepared, which, when administering in or applying onhuman or animal organism can be useful in preventing or controlling thecell damages cause by the above diseases thus relieving or eliminatingthe pathologic condition of the organism.

These compositions can be prepared by methods known per se in thepreparation of cosmetics and pharmaceutical compositions, by mixing theactive material and the corresponding carriers and/or auxiliaries. Thecompositions generally contain 0.5 to 99.5% by weight active compound.The amount of active material in the composition is determined by thenature and seriousness of disease, the age of patient and the mode oftreatment. The hydroxylamine derivatives of the formula (I) and (II) canbe formulated to compositions to be used orally and parenterally as wellas topically.

The daily dosis of the active compound is about 10 to 500 m/kg,preferably 20 to 100 mg/kg, which, especially in case of oralcompositions, is distributed to 2-3 administration.

For purposes of oral administration, the compositions are formulatedinto dragée, granulate, if desired, solution or suspension. Parenteralcompositions include aqueous suspensions and sterile injectablesolutions, while rectal administration forms are, among others,suppositories, and topical forms include ointments, cremes, emulsionsand gels.

For preparing tablets, the active ingredient is mixed with suitablecarriers, such as starch gelatin, lactose, magnesium stearate, talc,gumiarabicum and silicagel, the mixture is granulated and pressed intotablets.

In the preparation of dragées, a mixture similar to the above isprepared from the active ingredient and auxiliaries, the mixture isgranulated, the granulate is pressed into a core, which is then coatedwith sugar, e.g. by using a sugar-containing aqueouspolyvinylpirrolidone solution.

For preparing capsule forms, the active ingredient is mixed withauxiliaries, such as starch, talc, silica, microcrystalline cellulose,and the mixture is filled into hard or soft gelatin capsules.

These oral compositions may be completed with absorption promoting orretarding additives.

Syrups or elixirs or drops can be prepared by using, besides the activeingredient, sweeteners, methyl- or propyl-paraben and, if desired,tasting additives, by mixing the aqueous solution of the activeingredient therewith.

For rectal administration, suppositories can be prepared by using thesuitable auxiliaries, such as cocoa butter or polyethylene glycol.

Compositions suitable for parenteral administration can be theinjections, prepared by dissolving the active ingredient in sterileisotonic saline solution, or aqueous suspensions, which can be preparedby using suitable dispersing and wetting agents, such as propyleneglycol or butylene glycol.

The cremes and ointments for topical use can be prepared by usingprimary or secondary alcohols, such as cetyl alcohol, stearyl alcohol,glycerin, natural fats and oils, such as olive oil, wheat germ oil,lanolin, longer hydrocarbons, such as vaseline as well as cellulosederivatives. These compositions may also contain preservatives, such asmethyl-p-hydroxy benzoate.

The composition for use as cosmetics or medical cosmetics can beprepared in a similar way. Preferably, the lipophylic components aremixed, and the water-soluble components are dissolved in water,optionally by slight warming. If desired, the pH of the latter isadjusted to the suitable value and the emulsion thus obtained is stirreduntil cooling. The active ingredient is added to the mixture of themixture thus obtained in the form of aqueous solution.

The pharmaceutical and cosmetical compositions, which contain the novelhydroxylamine derivatives described in details under 4.1, in thisspecification can be prepared according to the above processes as well.These compositions form also an object of the invention.

One embodiment of the pharmaceutical and cosmetical compositionsaccording to the invention contains hydroxylamine derivatives accordingto the formula (I) in an amount of 0.5 to 99.5% by weight together withcarriers and auxiliaries generally used in such compositions, wherein

X is halo, preferably chloro or bromo,

Z is chemical bond, and

a1) A is a group of the formula (a), wherein Y¹ is halo, alkoxy,haloalkyl or nitro and n is 1, 2 or 3, or an O-containing heteroarylgroup, preferably furyl, S-containing heteroaryl, preferably thienyl, oran N-containing heteroaromatic group optionally condensed with a benzenering, or the N—C₁₋₄ alkyl quaternary derivative or N-oxide thereof,preferably piridyl, quinolyl or isoquinolyl,

R is a group of the formula (b), wherein R⁵ and R⁶, independently fromeach other are H, straight or branched alkyl, preferably C₁₋₇ alkyl orcycloalkyl, or R⁵ and R⁶ together with the N-atom adjacent thereto forma 3 to 7-membered, preferably 5 to 7-membered saturated heterocyclicring, Y⁶ is —OR², wherein R⁷ is H or acyl, preferably unsubstituted orsubstituted alkylcarbonyl, arylcarbonyl or aminoacyl, k is 1, 2 or 3 andm is 1, 2 or 3, or an N—C₁₋₄ alkyl quaternary derivative or N-oxidethereof, with the proviso, that when A is piridyl or naphtyl, or a groupof the formula (a) wherein Y¹ is halo or alkoxy, then R⁷ is other thanH, or

a2) A is a group of the formula (c),

R is a group of the formula (d) and the optional substituents Y² and Y³from which at least one must be present in the molecule, is oxygen orC₁₋₄ alkyl, and k is 1, 2 or 3 and m is 1, 2 or 3, and when the compoundis a mono- or bivalent cation, the anion is one or two halide ion,preferably iodide, or

X is —NR¹R², wherein R¹ and R², independently from each other, are H,unsubstituted or substituted straight or branched alkyl, unsubstitutedor substituted aryl, preferably phenyl, unsubstituted or substitutedaralkyl, or R¹ and R² together with the N-atom adjacent thereto, form a3 to 7-membered, preferably 5 to 7-membered heterocyclic ring, which maycontain one or more additional hetero atom(s),

A is unsubstituted or substituted aryl, preferably phenyl, orunsubstituted or substituted aralkyl,

Z is oxygen or ═NR³, wherein R³ is H or unsubstituted or substitutedalkyl, and

R is a group of the formula (b), wherein R⁵ and R⁶, independently fromeach other are H, straight or branched alkyl, preferably C₁₋₄ alkyl orcycloalkyl, or R⁵ and R⁶ together with the N-atom adjacent thereto forma 3 to 7-membered, preferably 5 to 7-membered saturated heterocyclicring, Y⁶ is H or —OR⁷, wherein R⁷ is H or acyl, preferably unsubstitutedor substituted alkylcarbonyl or arylcarbonyl, k is 1, 2 or 3 and m is 1,2 or 3, or

X is —OQ, wherein Q is unsubstituted or substituted alkyl or aralkyl,

Z is oxygen and

R is a group of the formula (b), wherein R⁵ and R⁶, independently fromeach other are H, straight or branched alkyl, preferably C₁₋₄ alkyl orcycloalkyl, or R⁵ and R⁶ together with the N-atom adjacent thereto forma 3 to 7-membered, preferably 5 to 7-membered saturated heterocyclicring, Y⁶ is H or —OR⁷, wherein R⁷ is H or acyl, preferably unsubstitutedor substituted alkylcarbonyl, arylcarbonyl or aminoacyl, k is 1, 2 or 3and m is 1, 2 or 3, or

A is unsubstituted or substituted aryl, preferably phenyl or anN-containing heteroaromatic group, preferably piridyl or an S-containingheteroaromatic group,

Z is a chemical bond.

X is —OQ, wherein Q is C₁₋₄ alkyl, and

R is a group of the formula (b), wherein R⁵ and R⁶, independently fromeach other are H, straight or branched alkyl, preferably C1-.J alkyl orcycloalkyl, or R⁵ and R⁶ together with the N-atom adjacent thereto forma 3 to 7-membered, preferably 5 to 7-membered saturated heterocyclicring, Y⁶ is H, k is 1, 2 or 3 and m is 1, 2 or 3.

Another group of the pharmaceutical and cosmetical compositionsaccording to the invention includes those which, together withpharmaceutically and cosmetically acceptable carriers and/or auxiliariescontain in an amount of about 0.5 to 99.5% by weight a hydroxylaminederivative of the formula (I) or the salts and/or the optically activestereoisomers thereof, wherein

X is —NR¹R², wherein R¹ and R², independently from each other, are H orunsubstituted or substituted straight or branched alkyl, preferably C₁₋₆alkyl or cycloalkyl or, R¹ and R² together with the N-atom adjacentthereto form a 3-7-membered, preferably 5 to 7-membered saturated heteroring,

A is unsubstituted or substituted aralkyl, preferably phenylalkylsubstituted with one or more alkoxy, preferably C₁₋₄ alkoxy,unsubstituted phenyl or phenyl substituted with one or more halo, alkylor haloalkyl, acylamino or nitro, or unsubstituted or substitutedN-containing heteroaromatic group which is optionally condensed with abenzene ring, preferably pirrolyl, piridyl, isoquinolyl or quinolyl, oran S-containing heteroaryl group, preferably thienyl, wherein theheteroaryl groups may have one or more substituents, preferably one ormore alkyl, preferably C₁₋₄ alkyl,

Z is a chemical bond, and

R is a group of the formulae (e), wherein R⁵ and R⁶, independently fromeach other are H, straight or branched alkyl, preferably C₁₋₄ alkyl orcycloalkyl, or R⁵ and R⁶ together with the N-atom adjacent thereto forma 3 to 7-membered, preferably 5 to 7-membered saturated heterocyclicring which may contain additional hetero atoms and may havesubstituent(s), preferably C₁₋₄ alkyl, Y⁴ is H or unsubstituted orsubstituted C₁₋₄ alkyl, Y⁵ is H, unsubstituted or substituted C₁₋₄ alkylor —OR⁷, wherein R⁷ is H or acyl, k is 1, 2 or 3 and m is 1, 2 or 3,with the proviso that

when A is unsubstituted phenyl or phenyl substituted with halo or alkoxyor phenylalkyl substituted with alkoxy or a piridyl group and R⁷ is H,at least one of R¹ and R² is other than H, and

when A is unsubstituted phenyl or phenyl substituted with halo or alkoxyor phenylalkyl substituted with alkoxy or a piridyl group, and R¹ and R²are both H, R⁷ is other than H.

Another group of the pharmaceutical and cosmetical compositionsaccording to the invention includes those which, together withpharmaceutically and cosmetically acceptable carriers and/or auxiliariescontain in an amount of about 0.5 to 99.5% by weight a hydroxylaminederivative of the formula (II) or the salts and/or the optically activestereoisomers thereof, wherein

X is oxygen,

A is C₁₋₂₀ straight or branched alkyl, unsaturated or saturated aryl,preferably phenyl or haloalkyl-phenyl, unsubstituted or substitutedaralkyl, naphtyl or an N-containing heteroaromatic group, preferablypiridyl,

Z is a chemical bond,

R′ is H, C₁₋₄ alkyl or aralkyl, preferably phenylalkyl, and

R is a group of the formula (b), wherein R⁵ and R⁶, independently fromeach other are H, straight or branched alkyl, preferably C₁₋₄ alkyl orcycloalkyl, or R⁵ and R⁶ together with the N-atom adjacent thereto forma 3 to 7-membered, preferably 5 to 7-membered saturated heterocyclicring, Y⁶ is H or —OR⁷, wherein R⁷ is H, k is 1, 2 or 3 and m is 1, 2 or3, with the proviso that when A is other than alkyl and R′ is H, Y⁶ isH, or

X is ═NR⁴, wherein R⁴ is H, unsubstituted or substituted alkyl orunsubstituted or substituted aryl, preferably phenyl, or unsubstitutedor substituted aryl, preferably phenylalkyl,

A is unsubstituted or substituted alkyl or unsubstituted or substitutedaryl, preferably phenyl or substituted phenyl, or unsubstituted orsubstituted aralkyl, preferably phenylalkyl, or cycloalkyl,

Z is a chemical bond, oxygen or ═NR³, wherein R³ is H or unsubstitutedor substituted alkyl,

R′ is unsubstituted or substituted alkyl or unsubstituted or substitutedaryl, preferably phenyl, or unsubstituted or substituted aralkyl,preferably phenylalkyl, and

R is a group of the formula (b), wherein R⁵ and R⁶, independently fromeach other are H, straight or branched alkyl, preferably C₁ alkyl orcycloalkyl, or R⁵ and R⁶ together with the N-atom adjacent thereto forma 3 to 7-membered, preferably 5 to 7-membered saturated heterocyclicring, Y⁶ is H or —OR⁷, wherein R⁷ is H or acyl, preferably unsubstitutedor substituted alkylcarbonyl or arylcarbonyl, k is 1, 2 or 3 and m is 1,2 or 3, or

X is oxygen,

A is unsubstituted or substituted alkyl, unsubstituted or substitutedaralkyl, preferably phenylalkyl,

Z is oxygen,

R′ is alkyl or aralkyl, preferably phenylalkyl,

R is a group of the formula (b), wherein R⁵ and R⁶, independently fromeach other are H, straight or branched alkyl, preferably C₁₋₄ alkyl orcycloalkyl, or R⁵ and R⁶ together with the N-atom adjacent thereto forma 3 to 7-membered, preferably 5 to 7-membered saturated heterocyclicring, Y⁶ is H or —OR⁷, wherein R⁷ is H or acyl, preferably unsubstitutedor substituted alkylcarbonyl or arylcarbonyl, k is 1, 2 or 3 and m is 1,2 or 3, or

X is oxygen,

Z is ═NH, and

d1) A is unsubstituted or substituted alkyl, cycloalkyl, unsubstitutedor substituted aralkyl, preferably phenylalkyl, unsubstituted phenyl orphenyl substituted with halo, haloalkyl, alkoxy or nitro,

R′ is alkyl or aralkyl, preferably phenylalkyl, and

R is a group of the formula (b), wherein R⁵ and R⁶, independently fromeach other are H, straight or branched alkyl, preferably C₁₋₄ alkyl orcycloalkyl, or R⁵ and R⁶ together with the N-atom adjacent thereto forma 3 to 7-membered, preferably 5 to 7-membered saturated heterocyclicring, Y⁶ is H or —OH, k is 1, 2 or 3 and m is 1, 2 or 3, or

d2) A is a group of the formula (a) wherein Y′ is haloalkyl, preferablytrifluoromethyl and n is 1, 2 or 3,

R is H and

R is a group of the formula (b), wherein R⁵ and R⁶, independently fromeach other are H, straight or branched alkyl, preferably C₁₋₄ alkyl orcycloalkyl, or R⁵ and R⁶ together with the N-atom adjacent thereto forma 3 to 7-membered, preferably 5 to 7-membered saturated heterocyclicring, Y⁶ is H or —OH, k is 1, 2 or 3 and m is 1, 2 or 3.

Another group of the pharmaceutical and cosmetical compositionsaccording to the invention includes those which, together withpharmaceutically and cosmetically acceptable carriers and/or auxiliariescontain in an amount of about 0.5 to 99.5% by weight a hydroxylaminederivative of the formula (I″) or the salts and/or the optically activestereoisomers thereof, wherein

A is unsubstituted phenyl or phenyl substituted with halo or nitro or anN-containing heteroaryl group; preferably piridyl,

R¹ is H and

R″ is an ω-aminoalkyl group which may be mono- or disubstituted, whereinthe alkyl chain contains 1 to carbon atoms and the amino-substituents,independently from each other are one or two straight or branched alkylor cycloalkyl, or wherein the two amino-substituent together with theN-atom adjacent thereto form a 3 to 7-membered, preferably 5 to7-membered heterocyclic ring or the N—C₁₋₄ alkyl quaternary derivativethereof, with the proviso that

when A is piridyl, R″ is other than 1-piperidinylmethyl.

The embodiments of the invention are illustrated in the followingexamples more in details. It should be understood, however, that thescope of protection is not limited to the specific embodiments set forthin the Examples.

CHEMICAL AND COMPOSITION EXAMPLES Example 1N-[2-hydroxy-3-(1-piperidinyl)propoxy]-2-thiophenecarboximidoyl chloridemonohydrochloride

5.0 g (15.6 mmol) ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-2-thiophenecarboximidamidemonohydrochloride (Example 44) was dissolved in 19 ml of water, then 6.1ml of concentrated hydrochloric acid was added. The solution was cooledto −5° C., then a cold solution of 4.4 g (63.8 mmol) of sodium nitritein 2.4 ml of water was added dropwise. Throughout the reaction theinternal temperature was maintained at 0° C. When addition was completedthe mixture was stirred for a further one hour. Cold benzene (60 ml) wasadded and the mixture was made alkaline with slow addition of a coldsolution of 3.2 g (80 mmol) of sodium hydroxide in 45 ml of water. Theorganic phase was separated and washed successively with 20 ml portionsof water until the pH<9 (3-5 times). The organic solution was dried overanhydrous sodium sulfate, treated with charcoal, filtered and evaporatedin vacuum (t<45° C.) to give 2.6 g of oil. This residue was dissolved in5 ml of isopropyl alcohol and acidified (pH 2) with isopropyl alcoholcontaining dry hydrochloric acid. The product was crystallized fromn-hexane to give off-white material.

Yield: 2.0 g (38%)

Mp.: 115-123° C.

Following the process described in the previous example the followingcompounds were prepared:

Example 2N-[2-hydroxy-3-(1-piperidinyl)propoxy]-1-isoquinolinecarboximidoylchloride monohydrochloride

Starting material: Example 46

Yield: 48%

Mp.: 168-172° C.

IR (KBr): 3425, 3128, 2947, 2866, 2650, 2540, 1622, 1597, 1556, 1452,1385, 1364, 1329, 1296, 1281, 1240, 1117, 1092, 1024, 1015, 978, 953,903, 881, 795, 743, 718, 658, 559 cm⁻¹

Example 3N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-quinolinecarboximidoyl chloride(Z)-2-butenedioate (1:1)

Starting material: Example 42

In this case the final product was isolated at the end of the work-upprocedure by dissolving the crude base in acetone, and adding anequivalent amount of maleic acid.

Yield: 67%

Mp.: 159-162° C.

IR (KBr): 3427, 3019, 2947, 2886, 2689, 1583, 1477, 1450, 1352, 1293,1221, 1194, 1132, 1072, 1045, 939, 919, 872, 833, 754, 650, 557 cm⁻¹

Example 4N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-nitro-benzenecarboximidoylchloride monohydrochloride

Starting material: Example 40.

Yield: 58%

Mp.: 185-1.89

Example 5N-[2-hydroxy-3-(1-piperidinyl]propoxy)-4-nitro-benzenecarboximidoylchloride monohydrochloride

Starting material: Example 43

Yield: 47%

Mp.: 180-182° C.

IR (KBr) 3331, 2953, 2853, 2735, 2654, 2577, 2548, 1605, 1568, 1516,1456, 1348, 1261, 1165, 1119, 1072, 1059, 1007, 960, 933, 862, 849, 754,719, 690, 673, 627, 581, 550, 478 cm⁻¹

Example 6N-[2-hydroxy-3-(1-piperidinyl)propoxy]-2-nitro-benzenecarboximidoylchloride monohydrochloride

Starting material: Example 45

Yield: 50%

Mp.: 159-162° C.

IR (KBr): 3298, 2983, 2932, 2746, 1593, 1574, 1535, 1445, 1391, 1354,1317, 1288, 1242, 1198, 1117, 1092, 1069, 1020, 968, 947, 914, 852, 793,756, 708, 577 cm⁻¹

Example 7 N-[2-hydroxy-3-(1-piperidinyl)propoxy]-2-furanecarboximidoylchloride monohydrochloride Procedure:

1-Chloro-2-hydroxy-3-(1-piperidinyl)-propane [J. Org. Chem. 33(2) p.523-30 (1968)] (3.0 g, 116.9 mmol) was dissolved in water (1.8 ml).Solid NaOH (1.19 g, 29.8 mmol) was added, and the mixture was stirred atroom temperature for 1 hour. N-Hydroxy-2-furanecarboximidamide (1.92 g,15.2 mmol) was added, and the mixture was kept on stirring at roomtemperature overnight. Concentrated HCl (2.1 ml) was added to adjust thepH to approx. 4, and the solution was evaporated in vacuum to dryness.

The residue (5.4 g) was dissolved in cc. HCl (37 ml), cooled to 0-5° C.,and an aqueous solution of NaNO₂ (5.6 g, 80 mmol in 23 ml water) wasadded dropwise in 30 min. The solution was made alkaline then byaddition of 2N NaOH solution (102 ml) to pH=10, and extracted with ethylacetate (2×130 ml). The combined organic phases were washed with water,dried over anh. Na₂SO₄ and evaporated. The residue (2.0 g) wasredissolved in a small volume of ethyl acetate (20 ml) and the productwas precipitated by addition of isopropanolic HCl solution (3.2 N, 3ml). The obtained white precipitate was filtered, washed, and finallyrecrystallized from isopropanol.

Yield: 11%

Mp.: 139-141° C.

IR (KBr): 3427, 3267, 3094, 2955, 2922, 2964, 2745, 1637, 1584, 1479,1452, 1391, 1319, 1281, 1259, 1157, 1117, 1074, 1024, 999, 980, 943,887, 854, 843, 743, 710, 596 cm⁻¹

Following the process described in the previous example the followingcompound was prepared:

Example 8 N-[2-hydroxy-3-(1-piperidinyl)propoxy]-4-pyridinecarboximidoylchloride (Z)-2-butenedioate (1:1)

In this case the final product was isolated at the end of the work-upprocedure by dissolving the crude base in acetone, and adding anequivalent amount of maleic acid.

Yield: 25%

Mp.: 165.5-169° C.

Example 9N-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-3-trifluoromethylbenzene-carboximidoylchloride monohydrochloride Procedure:

50 g (0.245 mol) of m-trifluoromethyl-benzamidoxime and 33.7 g (0.6 mol)of potassium hydroxide was dissolved in a mixture of dimethyl sulphoxideand 170 ml of water, and the mixture was cooled to 0° C. 48 ml (0.6 mol)of epichlorohydrine was added, and the reaction mixture was stirred at0° C. for 5 hours, then kept in a refrigerator overnight. Next day 250ml of water was added, and the mixture was extracted with ethyl acetate(4×250 ml). The combined organic phases were washed with water, dried,treated with charcoal and evaporated to dryness, to yieldm-trifluoromethyl-N-(2,3-epoxypropoxy)-benzamidine, as a colorless oil.

Yield: 61 g (96%)

To the obtained oil 400 ml of 18% of hydrochloric acid solution and 60ml of ether were added, and the mixture was cooled to −5° C., whilestirring. 17.4 g (0.25 mol) of sodium nitrite, dissolved in 60 ml ofwater was added slowly in 40 min., and the reaction mixture was stirredfor another 20 minutes. The mixture was extracted then with ether (2×160ml), and the combined organic phases were washed with water twice. Tothe ethereal solution 340 ml of 20% of sodium hydroxide solution wasadded, and the two-phase system was refluxed for 1 hour, while stirring.The phases were then separated, the organic layer was washed with brineuntil neutral, dried and evaporated to dryness to givem-trifluoromethyl-N-(2,3-epoxypropoxy)-benzimidoyl chloride, as acolorless oil.

Yield: 30.5 g (45%)

A mixture of 1.19 g (4.2 mmol)N-[(2,3-epoxy)propoxy]-3-trifluoromethyl-benzenecarboximidoyl chlorideand 0.89 ml (8.5 mmol) of tert-butylamine in 12 ml of isopropyl alcoholwas refluxed for 2 hours. Solvent was removed under reduced pressure.The residue was dissolved in ethyl acetate, and 0.98 ml of methanolichydrogen chloride solution (4.3 N) was added and the mixture wasconcentrated to small volume under vacuum, then diluted with ether. Theprecipitate that formed was recovered, washed with cold ether and dried.

Yield: 0.48 g (32%)

Mp.: 150-153° C.

IR(KBr): 3423, 3233, 2978, 2880, 2784, 1620, 1570, 1479, 1441, 1400,1383, 1340, 1238, 1167, 1128, 1101, 1072, 1038, 982, 930, 897, 804, 787,714, 694 cm⁻¹

Following the process described in the previous example the followingcompounds were prepared:

Example 10N-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]-3-trifluoromethyl-benzene-carboximidoylchloride monohydrochloride

Yield: 30%

Mp.: 105-108° C.

IR (KBr): 3358, 2984, 2883, 2804, 1595, 1441, 1383, 1335, 1238, 1184,1171, 1121, 1099, 1074, 1011, 995, 947, 906, 891, 798, 779, 696, 681,567 cm⁻¹

Example 11N-[3-(cyclohexylamino)-2-hydroxypropoxy]-3-trifluoromethyl-benzenecarboximidoylchloride monohydrochloride

Yield: 35%

Mp.: 147-149.5° C.

IR (KBr): 3381, 2951, 2860, 2820, 1580, 1439, 1344, 1246, 1161, 1126,1099, 1074, 1003, 986, 932, 903, 872, 802, 787, 716, 692, 681, 648 cm⁻¹

Example 12N-[3-(diethylamino)-2-hydroxypropoxy]-3-trifluoromethyl-benzenecarboximidoylchloride monohydrochloride

Yield: 21%

Mp.: 121-128° C.

IR(KBr): 3425, 3289, 2951, 2667, 1818, 1443, 1337, 1238, 1178, 1115,1078, 1049, 997, 910, 804, 781, 696, 683 cm⁻¹

Example 13N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-trifluoromethyl-benzenecarboximidoylchloride monohydrochloride

Yield: 13%

Mp.: 119-123° C.

IR (KBr): 3366, 2937, 2854, 2737, 2673, 2538, 1616, 1570, 1439, 1404,1337, 1290, 1236, 1199, 1165, 1129, 1101, 1074, 1030, 984, 972, 933,901, 829, 804, 788, 717, 699, 685, 646 cm⁻¹

Example 14N-[2-hydroxy-3-(piperidinyl-1-oxide-1-yl)propoxy]-N′-oxy-3-pyridinecarboximidoylchloride Procedure:

To a solution ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloride(5.0 g; 17.1 mmol) in chloroform (50 ml) m-chloroperbenzoic acid (7.0 g;40 mmol) was added in small portions, and the mixture was stirred atroom temperature for 2 hours. The solvent was removed, the residue wasdissolved in 80 ml of ethyl acetate, extracted with water, dried andevaporated. The obtained oily product was finally crystallized withacetone to give the product as an off-white solid.

Yield: 2.21 g (6.7 mmol; 40%)

Mp.: 140-142° C.

IR (KBr): 3437, 3071, 2943, 2880, 2590, 1801, 1578, 1475, 1454, 1433,1375, 1294, 1259, 1194, 1165, 1121, 1088, 1043, 1011, 995, 924, 905,888, 845, 808, 710, 671, 554, 513, 413 cm⁻¹

Example 15N-[2-hydroxy-3-(piperidin-1-oxide-1-yl)propoxy]-3-pyridinecarboximidoylchloride Procedure:

To a solution ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloride(2.0 g; 6.8 mmol) in chloroform (20 ml) m-chloroperbenzoic acid (1.6 gof 70% purity; 6.5 mmol) was added, and the mixture was stirred at roomtemperature for 30 minutes. The solution was made alkaline with 10% ofsodium hydroxide solution, then separated, and the organic layer waswashed with brine, dried and evaporated. The solid residue wasrecrystallized with ethyl acetate, the precipitate was filtered off,washed and dried, to give the product as a white solid.

Yield: 1.03 g (48%)

Mp.: 127-130° C.

IR (KBr): 3454, 2988, 2945, 2880, 2585, 1585, 1512, 1479, 1443, 1416,1393, 1350, 1331, 1289, 1183, 1134, 1072, 1051, 1030, 997, 953, 939,879, 847, 808, 702, 519, 417 cm⁻¹

Example 16N′-[2-hydroxy-3-(1-methyl-1-piperidinium-1-yl)propoxy]-N-methyl-pyridinium-3-carboximidoylchloride diiodide Procedure:

A mixture ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloride(1.0 g; 3.4 mmol) and 1.2 ml (20 mmol) of methyl iodide was refluxed inacetone (10 ml) under nitrogen for 2 hours. The resulting dark yellowprecipitate was filtered off, and washed with acetone to give the crudeproduct (1.8 g) which was then recrystallized from 20 ml of ethanol.

Yield: 1.2 g (60%)

Mp.: 153-157° C.

IR (KBr): 3462, 3406, 3317, 3040, 2941, 2878, 2831, 1729, 1636, 1589,1504, 1462, 1378, 1350, 1290, 1209, 1171, 1121, 1069, 1047, 1030, 1001,941, 897, 868, 818, 706, 673, 635, 589 cm⁻¹

Example 17N-[2-acetoxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloride(Z)-2-butenedioate (1:1) Procedure:

1.48 g (5.0 mmol) ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridine-carboximidoyl chloridewas dissolved in 5 ml of acetic anhydride. The temperature of thereaction was raised up to 40° C. After 30 minutes at room temperaturethe solvent was completely removed in vacuum, the residue was dissolvedin 30 ml of diethyl ether, treated with charcoal, filtered and thesolvent was removed under reduced pressure to give 1.74 g oforange-colored oil.

The residue was dissolved in 10 ml of acetone, and a solution of 0.6 g(5.17 mmol) of maleic acid in 10 ml of acetone was added. Thecrystalline product was removed by filtration and washed with acetone togive 1.43 g off-white material. Recrystallization, with decolorization,from 9 ml of isopropyl alcohol produced the title compound.

Yield: 1.22 g (54%)

Mp.: 143-144° C.

Example 18(S)—N-[2-[2-®-(1,1-dimethylethyloxycarbonylamino)-3-phenylpropionyloxy]-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride (Z)-2-butenedioate (1:1) Procedure:

6.7 g (25.5 mmol) of N-(tert-butoxycarbonyl)-D-phenylalanine wasdissolved in 50 ml of dichloromethane. The solution was cooled to 0° C.,and 4.0 ml of triethylamine and then 2.5 ml (26 mmol) of ethylchloroformate was added dropwise. The mixture was stirred for 20 minutesat 0° C., then a solution of 7.5 g (26 mmol) ofN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridinecarboximidoyl chloridein 50 ml of dichloromethane was added in 30 minutes. The reactionmixture was stirred at room temperature for 1 hour. The solution wasextracted first with 10% acetic acid (2×100 ml), then with water, driedwith anhydrous sodium sulfate, and evaporated to dryness. The residue(10.7 g) was dissolved in 71 ml of acetone and 1.53 g (13 mmol) ofmaleic acid was added. The resulting solid was filtered off and washedwith acetone.

Yield: 4.0 g (6.0 mmol; 23%)

Mp.: 146.5-148° C.

[α]_(D)=+21.5° (c=1, MeOH)

IR(KBr): 3393, 2978, 1744, 1697, 1582, 1518, 1468, 1454, 1420, 1381,1358, 1313, 1290, 1256, 1213, 1169, 1126, 1099, 1084, 1045, 1016, 930,908, 870, 750, 690, 575 cm⁻¹

Following the process described in the previous example the followingcompound was prepared:

Example 19(R)—N-[2-[2-(S)-(1,1-dimethylethyloxycarbonylamino)-3-phenylpropionyloxy]-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride (Z)-2-butenedioate (1:1) Yield: 25%

This compound has the same physical data (Mp.; IR) as written in Example18.

[α]_(D)=−23.6° (c=1, MeOH)

Example 20N-[2-benzoyloxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidamide(Z)-2-butenedioate (1:1) Procedure:

20.9 g (75.0 mmol) ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridine-carboximidamide [Hung.Pat. 177.578 (1976)] was dissolved in 300 ml of benzene. To thissolution 150 ml of 1 N sodium hydroxide solution was added, followed bydropwise addition of 19.5 ml (168 mmol) of benzoyl chloride. Afterstirring the mixture intensively for 2 hours, 7.1 g (67 mmol) of sodiumcarbonate and a further portion of benzoyl chloride (9.75 ml; 84 mmol)was added, and the stirring was continued overnight. The phases werethen separated, the organic layer was extracted with 1 N sodiumhydroxide solution and water, dried and evaporated to dryness. Theresidue (41 g oil) was dissolved in 150 ml of acetone, and 8.7 g (75mmol) maleic acid was added. The obtained precipitate was filtered off,washed with acetone, and dried.

Yield: 29.1 g (78%)

Mp.: 194-195° C.

Following the process described in the previous example the followingcompound was prepared:

Example 21N-[2-benzoyloxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride (Z)-2-butenedioate (1:1)

Starting material: U.S. Pat. No. 5,147,879 (1992)

Yield: 64%

Mp.: 134-136° C.

IR(KBr): 2955, 2939, 2517, 1718, 1583, 1477, 1452, 1410, 1370, 1354,1317, 1268, 1209, 1173, 1117, 1057, 1043, 998, 968, 939, 903, 870, 748,723, 714, 652, 582 cm⁻¹

Example 22N-[2-palmitoyloxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidamidemonohydrochloride Procedure:

14.7 g (52.8 mmol) ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridine-carboximidamide [Hung.Pat. 177.578 (1976)] was dissolved in 160 ml of chloroform. 7.7 ml (55mmol) of triethylamine was added, followed by dropwise addition of asolution of palmitoyl chloride (14.7 g; 56.5 mmol) in 85 ml ofchloroform. The mixture was stirred overnight at room temperature. Nextday further amount of 3.8 ml of triethylamine and 7.4 g ofpalmitoylchloride was added, and the stirring was continued for one moreday. The solution was extracted then with water, 5% acetic acid andwater, successively, dried over anh. sodium sulfate, and evaporated todryness.

The residue (28.2 g oil) was dissolved in ethyl acetate, and the productwas precipitated by addition of 30 ml of 1 N HCl/ethyl acetate. Thethick, white precipitate was filtered off, washed with ethyl acetate anddried.

Yield: 10.9 g (37%)

Mp.: 110-113° C.

Following the process described in the previous example the followingcompounds were prepared:

Example 23N-[2-palmitoyloxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride dihydrochloride

Starting material: U.S. Pat. No. 5,147,879 (1992)

Note: the reaction was carried out by refluxing.

Yield: 72%

Mp.: 69-73.5° C.

IR(KBr): 3425, 2922, 2853, 2648, 2544, 1742, 1632, 1468, 1416, 1377,1287, 1183, 1113, 1087, 1032, 984, 708, 675 cm⁻¹

Example 24N-[2-(2-furoyloxy)-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidamide(Z)-2-butenedioate (1:1)

Note: the product was isolated in the form of maleate salt.

Yield: 52%

Mp.: 167-171.5° C.

Example 25N-[2-(o-chlorobenzoyloxy)-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidamidemonohydrochloride

Note: the reaction was carried out by refluxing.

Yield: 50%

Mp.: 91-94° C.

Example 26N-[2-(p-methoxybenzoyloxy)-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidamidemonohydrochloride

Note: the reaction was carried out by refluxing.

Yield: 71%

Mp.: 152-155° C.

Example 27N-[2-(m-trifluoromethylbenzoyloxy)-3-(1-piperidinyl)propoxy]-3-pyridine-carboximidamidemonohydrochloride

Note: the reaction was carried out by refluxing.

Yield: 45%

Mp.: 144-147° C.

Example 28N-[2-(2-thenoyloxy)-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidamide(Z)-2-butenedioate (1:1)

Note: the reaction was carried out by refluxing, and the product wasisolated in the form of maleate salt.

Yield: 58%

Mp.: 168-176° C.

Example 29N-[2-acetoxy-3-[(1-piperidinyl)propoxy]-3-pyridinecarboximidamidemonohydrochloride Procedure:

2.5 g (9.0 mmol) ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidamide wasdissolved in 27 ml of chloroform, 1.6 g (16 mmol) of acetic anhydridewas added and stirred at room temperature for 1 hour. The reactionmixture was evaporated to dryness, and dissolved in isopropyl alcoholcontaining the equimolar quantity (9 mmol) of dry hydrogen chloride. Thesolution was cooled and the solid were filtered. Recrystallization fromisopropyl alcohol gave white crystalline compound.

Yield: 1.9 g (59%)

Mp.: 107° C.

Example 30N-[2-(3-pyridinecarbonyloxy)-3-(1-piperidinyl)]propoxy-3-pyridinecarboximidamide-(Z)-2-butenedioate(1:1) Procedure:

To a solution ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximid-amide (1.68g; 6 mmol) in dry pyridine 1.68 g (7.4 mmol) of nicotinic anhydride wasadded and kept at room temperature overnight. The mixture wasevaporated, the residue was dissolved in 30 ml of ethyl acetate,filtered, the filtrate was extracted with 10% NaHCO₃ solution, dried andevaporated. The obtained oil was dissolved in 20 ml of acetone, and 0.53g of maleic acid was added to result in precipitation. The product wasfiltered off and washed with acetone.

Yield: 1.84 g (61%)

Mp.: 157-160° C.

Example 31 N-[3-(1-piperidinyl)propoxy]-3-pyridinecarboximidamidedihydrochloride Procedure:

2.86 g (51.1 mmol) of potassium hydroxide was dissolved in 20 ml of abs.ethanol, then 6.45 g (47.0 mmol) N-hydroxy-3-pyridinecarboximidamide and7.7 g (47.7 mmol) 1-chloro-3-(1-piperidinyl)-propane were added andrefluxed for 9 hours. The solid was removed by filtration and thefiltrate was evaporated. The crude product was dissolved in 100 ml ofchloroform, washed with 1 N of sodium hydroxide solution then threetimes of water. The organic layer was dried over sodium sulfate,filtered and the solvent was evaporated under reduced pressure. Theresidue was dissolved in a small amount of abs. ethanol and isopropylalcohol containing dry hydrochloric acid was added (pH 2) to affordoff-white crystals.

Yield: 4.8 g (38%)

Mp.: 95-100° C. (dec.)

IR (KBr, base): 3422, 3294, 3107, 2984, 2937, 2870, 2818, 1649, 1616,1593, 1479, 1462, 1441, 1381, 1309, 1194, 1123, 1094, 1059, 1042, 982,910, 858, 816, 712, 559 cm⁻¹

Following the process described in the previous example the followingcompounds were prepared:

Example 32N-[3-(1-piperidinyl)propoxy]-3-trifluoromethyl-benzenecarboximidamidemonohydrochloride

Yield: 42%

Mp.: 116-119° C.

IR (KBr, base): 3412, 3082, 2949, 2874, 2827, 1655, 1485, 1447, 1383,325, 1283, 1171, 1121, 1094, 1072, 986, 920, 905, 808, 700, 677, 627cm⁻¹

Example 33N-[3-(1-piperidinyl)propoxy]-(3,4-dimethoxyphenyl)methanecarboximidamidedihydrochloride

Yield: 35%

Mp.: 207-209° C.

Example 34N-[2,2-dimethyl-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidamide

Yield: 38% (oil)

IR (KBr): 3323, 2935, 2888, 2785, 1637, 1477, 1393, 1360, 1157, 1111,1057, 995, 943, 860, 814, 789, 708, 627 cm⁻¹

Example 35N-[3-(4-methyl-1-piperazinyl)propoxy]-3-pyridinecarboximidamidemonohydrochloride

Yield: 23%

Mp.: 127-130° C.

IR (KBr): 3387, 2947, 2878, 2802, 1730, 1639, 1450, 1389, 1283, 1242,1194, 1150, 1083, 1015, 964, 933, 814, 710 cm⁻¹

Example 36 N-(3-(1-piperidinyl)propoxy)-3-nitro-benzenecarboximidamidemonohydrochloride

Yield: 51%

Mp.: 158-162° C.

Example 37 N-[3-(1-piperidinyl)propoxy]-benzenecarboximidamidedihydrochloride

Yield: 64%

Mp.: 207-209° C.

Example 38N-[2-hydroxy-3-[(1-piperidinyl)propoxy]-2,4,6-trimethyl-benzenecarboximidamide

Yield: 44%

Mp.: 199-201° C.

IR (KEr): 3410, 3103, 2943, 2912, 2814, 2791, 1634, 1582, 1441, 1383,1350, 1321, 1304, 1254, 1204, 1146, 1111, 1099, 1065, 993, 878, 851,785, 754, 525 cm⁻¹

Example 39N-[2-hydroxy-3-[(1-piperidinyl)propoxy]-4-acetamino-benzenecarboximidamidemonohydrochloride

Yield: 25%

Mp.: 133-137° C.

Example 40N-[2-hydroxy-3-[(1-piperidinyl)propoxy]-3-nitro-benzenecarboximidamidedihydrochloride

Yield: 38%

Mp.: 190-193° C.

Example 41N-[2-hydroxy-3-[(1-piperidinyl)propoxy]-2-(1,5-dimethyl)-pyrrolcarboximidamidemonohydrochloride

Yield: 20%

Mp.: 144-147° C.

IR (KBr, base): 3458, 3369, 2930, 2849, 1622, 1587, 1502, 1468, 1437,1396, 1354, 1323, 1279, 1254, 1200, 1157, 1115, 1078, 1042, 988, 962,930, 870, 856, 758, 737, 694, 609 cm⁻¹

Example 42N-[2-hydroxy-3-[(1-piperidinyl)propoxy]-3-quinolinecarboximidamidedihydrochloride

Yield: 36%

Mp.: 210-211° C.

Example 43N-[2-hydroxy-3-[1-piperidinyl)propoxy]-4-nitro-benzenecarboximidamidedihydrochloride

Yield: 77%

Mp.: 184-189° C.

Example 44N-[2-hydroxy-3-(1-piperidinyl)propoxy]-2-thiophenecarboximidamidedihydrochloride

Yield: 73%

Mp.: 157-170° C.

IR (KBr): 3280 (b), 2940, 1655, 1420, 1120, 1018, 1002, 857, 710 cm⁻¹

Example 45N-[2-hydroxy-3-(1-piperidinyl)propoxy]-2-nitro-benzenecarboximidamidedihydrochloride

Yield: 47%

Mp.: 200-208° C.

IR (KBr): 3300 (b), 2960, 1670, 1535, 1347, 1155, 1020, 1002, 860, 800,753 cm⁻¹

Example 46N-[2-hydroxy-3-(1-piperidinyl)propoxy]-1-isoquinolinecarboximidamidedihydrochloride

Yield: 56%

Mp.: 208-216° C.

Example 47N-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-3-trifluoromethyl-benzenecarboximidamidedihydrochloride Procedure:

A mixture of 21.0 g (80.8 mmol) ofm-trifluoromethyl-N-(2,3-epoxypropoxy)-benzamidine (Example 9/a), 105 mlof tert-butylamine, 210 ml of ether and 84 ml of 4 N sodium hydroxidesolution was refluxed for 5 hours. The phases were separated, theethereal layer was washed with brine, dried and evaporated to dryness.The resulting oil (25.8 g) was dissolved in 250 ml of acetone, treatedwith charcoal, then 39 ml of 4 N HCl/ethyl acetate solution was added,while stirring, resulting in precipitation of a white solid, which wasfiltered off and washed with acetone.

Yield: 22.8 g (70%)

Mp.: 186-192° C. (dec.)

IIR (KBr): 3418, 2984, 2785, 2625, 2527, 2401, 1664, 1585, 1487, 1437,1381, 1329, 1173, 1155, 1130, 1078, 905, 874, 820, 692, 642, 594 cm⁻¹

Example 48N-[2-hydroxy-3-(1-piperidinyl)propoxy]-N′-butyl-3-pyridinecarboximidamidemonohydrochloride Procedure:

Preparation ofN-[2-[(2-tetrahydropyranyl)oxy)-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride (21.3 g, 71.4 mmol) ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridewas dissolved in 500 ml of chloroform, acidified with etherealhydrochloric acid solution to pH=3, then 32.6 ml (0.357 mol) of3,4-dihydro-2H-pyrane was added. The mixture was stirred at roomtemperature for 20 hours, washed three times with 200 ml portions of 2 Nsodium hydroxide solution and four times with the same amount of water.The organic phase was dried over sodium sulfate, filtered and evaporatedunder reduced pressure. The oily residue was dissolved in 600 ml ofethyl acetate and washed four times with 150 ml portions of a pH=5buffer solution. The organic solution was dried, filtered and evaporated

Yield: 24.5 g (90%)

A mixture of 3.7 g (9.68 mmol) ofN-[2-[(2-tetrahydropyranyl)oxy]-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride and 40 ml (0.41 mol) n-butylamine was refluxed for 3 hours. Theexcess of amine was evaporated in vacuum affording dark-brown oil, whichwas dissolved in 40 ml of ethanol containing 3.0 g of 4-toluenesulphonicacid and the mixture was heated at 60° C. for one hour. The solvent wasremoved under reduced pressure, the residue was made alkaline (pH 10)with 2 N of sodium hydroxide solution then extracted three times withchloroform. The organic solution was dried over sodium sulfate, filteredand the solvent was evaporated in vacuum. The dark oily residue waspurified by chromatography to give the pure base, which was dissolved in20 ml of ethanol and acidified with equivalent amount of dryhydrochloric acid dissolved in isopropyl alcohol to give the titlecompound as a pale-yellow crystalline solid.

Yield: 1.22 g (34%)

Mp.: 120-122° C.

IR (KBr, base): 3319, 3293, 3040, 2959, 2928, 2854, 2842, 2552, 1612,1580, 1450, 1427, 1399, 1333, 1315, 1221, 1196, 1171, 1126, 1103, 1051,1022, 964, 928, 899, 858, 829, 719, 692, 602 cm⁻¹

Following the process described in the previous example the followingcompound was prepared:

Example 49N-[2-hydroxy-3-(1-piperidinyl)propoxy]-N′-cyclohexyl-3-pyridinecarboximidamidemonohydrochloride

Yield: 0.89 g (24%)

Mp.: 130-134° C.

IR (KBr): 3280, 2935, 2853, 2640, 1720, 1619, 1551, 1514, 1452, 1404,1313, 1236, 1194, 1155, 1124, 1111, 1090, 1040, 978, 828, 735, 627 cm⁻¹

Example 50N-[2-hydroxy-3-(1-piperidinyl)propoxy-N′-(1,1-dimethylethyl)-benzenecarboximidamideProcedure:

Into a solution of 0.92 g (5.2 mmol) of1-chloro-2-hydroxy-3-(1-piperidinyl)-propane in 2 ml of water 0.42 g(10.4 mmol) sodium hydroxide was added and stirred for one hour. To thismixture was added 1.0 g (5.2 mmol) ofN-hydroxy-N′-(1,1-dimethylethyl)-benzenecarboximidamide dissolved in 20ml of ethanol and refluxed for 4 hours. Solvent was evaporated and 50 mlof water was added and three times was extracted with 50 ml portions ofchloroform. The organic phase was dried over sodium sulfate, filteredand the solvent was evaporated in vacuum. The yellow oily residue wasslowly crystallized in refrigerator. The crystals were triturated withdiethyl ether and filtered off.

Yield: 0.55 g (31%)

Mp.: 134-137° C.

IR (KBr): 3427, 3254, 2929, 2853, 2814, 1739, 1603, 1510, 1445, 1391,1367, 1302, 1281, 1190, 1140, 1117, 1094, 1067, 1036, 993, 963, 922,841, 789, 716, 675 cm⁻¹

Example 51N-[2-hydroxy-3-(1-piperidinyl)propoxy-N′,N′-diethyl-3-pyridinecarboximidamidemonohydrochloride Procedure:

0.66 g (16.6 mmol) of sodium hydroxide was dissolved in 25 ml of abs.ethanol, then 1.61 g (8.3 mmol)N-hydroxy-N′,N′-diethyl-3-pyridinecarboximidamide and 1.48 g (8.3 mmol)1-chloro-2-hydroxy-3-(1-piperidinyl)-propane were added and refluxed for5 hours. Solvent was evaporated and 50 ml of water was added and threetimes was extracted with 50 ml portions of ethyl acetate. The organiclayer was dried over sodium sulfate, filtered and the solvent wasevaporated in vacuum. The yellow oily residue was purified bychromatography to give the pure base, which was 64 dissolved in 20 ml ofethyl acetate and acidified with equivalent amount of dry hydrochloricacid dissolved in ethyl acetate to give the title compound as a whitecrystalline solid.

Yield: 1.3 g (42%)

Mp.: 113-117° C.

Example 52 N-[2-hydroxy-3-(1-piperidinyl)propoxy]-hexadecanoicamidemonohydrochloride Procedure:

1.74 g (10 mmol) 1-aminooxy-2-hydroxy-3-(1-piperidinyl)propane wasdissolved in 20 ml of chloroform and cooled to 0°. A solution ofpalmitoylchloride (2.85 g; 10 mmol) in 10 ml of chloroform was addeddropwise in 10 min. After stirring the mixture for 15 minutes theobtained white precipitate was filtered off, washed with chloroform, anddried.

Yield: 3.2 g (71%)

Mp.: 147-150° C.

IR (KBr): 3242, 3090, 2951, 2916, 2849, 1730, 1653, 1520, 1472, 1439,1371, 1300, 1229, 1169, 1136, 1099, 1070, 1009, 993, 962, 928, 858, 760,719, 602, 471 cm⁻¹

Following the process described in the previous example the followingcompounds were prepared:

Example 53 N-[3-(1-piperidinyl)propoxyl]-3-trifluoromethyl-benzamide

Starting material: EP 365,364 (1990)

Yield: 69% (oil)

IR (KBr): 3425, 2941, 2864, 2775, 1674, 1614, 1566, 1520, 1483, 1441,1393, 1337, 1319, 1277, 1187, 1129, 1072, 922, 914, 750, 698, 650 cm⁻¹

Example 54N-[2-hydroxy-3-(1-piperidinyl)propoxy]naphthalene-1-carboxamide

Yield: 54%

Mp.: 104-107° C.

IR (KBr): 3375, 2934, 1641, 1593, 1564, 1439, 1340, 1325, 1113, 1026,941, 810, 779 cm⁻¹

Example 55 N-[2-hydroxy-3-(1-piperidinyl)propoxy]-N′-heptyl-ureaProcedure:

To the solution of 1.23 g (7.1 mmol) of1-aminooxy-2-hydroxy-3-(1-piperidinyl)-propane dissolved in 20 ml ofchloroform 1.0 g (7.1 mmol) of heptyl isocyanate was added and thereaction mixture was stirred for 20 hours. Solvent was evaporated invacuum and the residue was purified by chromatography to give purecolorless oil. White crystalline product was obtained by trituratingwith petroleum ether.

Yield: 81%

Mp.: 49-51° C.

Following the process described in the previous example the followingcompound was prepared:

Example 56 N-[2-hydroxy-3-(1-piperidinyl)propoxy]-N′-propyl-urea

Yield: 50% (oil)

IR (KBr): 3319, 2934, 2878, 2802, 1666, 1551, 1456, 1393, 1308, 11551092, 1040, 993, 889, 793 cm⁻¹

Example 57 N-cyclohexyl-N′[2-hydroxy-3-(1-piperidinyl)propoxy]-urea

Yield: 67%

Mp.: 108-110 C.°

IR (KBr): 3319, 3287, 3188, 2930, 2853, 2797, 1637, 1574, 1452, 1354,1331, 1300, 1101, 1098, 991 cm⁻¹

Example 58 N-hexyl-N′-[2-hydroxy-3-(1-piperidinyl)propoxy]-urea

Yield: 27%

Mp.: 50-52 C.°

IR (KBr): 3310, 2932, 2858, 2804, 1666, 1551, 1454, 1377, 1306, 1092,1040, 995, 791, 725, 604 cm⁻¹

Example 59N-(3-chlorophenol)-N′-[2-hydroxy-3-(1-piperidinyl)propoxy]-urea

Yield: 34%

Mp.: 117-118 C.°

IR (KBr): 3250, 2939, 2900, 1670, 1597, 1551, 1491, 1429, 1329, 1252,1119 972, 775, 718, 700 cm⁻¹

Example 60N-cyclohexyl-N′-[2-hydroxy-3-[N-cyclohexylcarbamoyl-N-(1,1-dimethylethyl)-amino]propoxy]-urea

Yield: 44%

Mp.: 151-152 C.°

IR (KBr): 3312, 2932, 2854, 1668, 1616, 1555, 1450, 1393, 1364, 1354,1252, 1220, 1130, 941, 891 cm⁻¹

Example 61 N-hexyl-W-[3-(1-piperidinyl)propoxy]-urea

Yield: 85% (oil)

IR (KBr): 3354, 2932, 2856, 2810, 2777, 1666, 1543, 1486, 1377, 1308,1155, 1134, 1076 cm⁻¹

Example 62 N-tert-butyl-N′-[(2-hydroxy-3-(1-piperidinyl)propoxy)]-urea

Yield: 38%

Mp: 71-73 C.°

IR (KBr): 3314, 2945, 2916, 1651, 1555, 1460, 1393, 1384, 1335, 1254,1111, 988, 903, 839, 781 cm⁻¹

Example 63N-(3-nitro-phenyl)-N′-[(2-hydroxy-3-(1-piperidinyl)propoxy)]-urea

Yield: 54%

Mp: 137-139 C.°

IR (KBr): 3281, 2943, 2818, 1672, 1607, 1560, 1529, 1486, 1437, 1354,1283, 1115, 802, 739 cm⁻¹

Example 645,6-Dihydro-5-(1-piperidinyl)methyl-3-(3-pyridyl)-4H-1,2,4-oxadiazineProcedure:

17.5 g (0.05 mole) ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidamidedihydrochloride was dissolved in 50 ml of thionyl chloride, boiled forone hour, then the mixture was evaporated to dryness. The residue wasdissolved in 300 ml of methanol, treated with charcoal and afterfiltration the solvent was evaporated in reduced pressure. The residuewas dissolved in the minimum amount of ethanol and refrigerated to yieldcrystallineN-[2-chloro-3-(1-piperidinyl)propoxy]-3-pyridine-carboximidamidedihydrochloride as intermediate compound.

Yield: 13.2 g (71%)

Mp.: 127-145° C.

13.2 g (35.7 mmol) ofN-[2-chloro-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidamidedihydrochloride was added to a solution of 16.5 g (143.5 mmole) ofpotassium tert-butoxide dissolved in 150 ml of tert-butanol. The mixturewas boiled for 6 hours, then evaporated in vacuum. 100 ml of 5% sodiumhydroxide solution was added and the mixture was extracted three timeswith 300 ml portions of ethyl acetate.

The organic layer was dried over sodium sulfate, filtered and evaporatedto dryness. The residue was triturated with diethyl ether to yield thetitle compound as white crystals.

Yield: 3.5 g (38%)

Mp.: 157.5-158° C.

Example 65N-[3-[1,1-dimethylethyl)amino]-2-hydroxypropoxy]-3-trifluoromethyl-benzamideProcedure:

1.3 ml (15.2 mmol) of epichlorohydrine was added to a solution of 1.6 ml(15.2 mmol) of tert-butylamine in 8 ml of ethanol during 10 minutes withstirring, keeping the temperature below 20° C., and allow to stand for 3days.

Separately, 0.8 g (14.3 mmol) of potassium hydroxide was dissolved in amixture of 20 ml of ethanol and 3 ml of water and into this solution3.42 g (15.2 mmol) of N-hydroxy-3-(trifluoromethyl)-benzamide potassiumsalt and the formerly prepared solution of epichlorohydrine andtert-butylamine was added. The reaction mixture was stirred and boiledfor 10 hours, then the solvent was evaporated. The residue wastriturated with 20 ml of dichloromethane and 10 ml of water, the organicphase was separated, washed with 5 ml of water and 5 ml of saturatedsodium chloride solution, dried over sodium sulfate, filtered andevaporated. The oily residue was crystallized in a mixture ofacetone-hexane to yield white powder as title compound.

Yield: 0.85 g (17.3%)

Mp.: 156-158° C.

IR (KBr): 2976, 2858, 1612, 1556, 1379, 1352, 1313, 1273, 1165, 1072,694 cm

Example 66Methyl-N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidate(Z)-2-butenedioate (1:1) Procedure:

11.4 g (38.2 mmol) ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridewas dissolved in 60 ml of abs. methanol, then 25 ml (0.1 mole) of 25%methanolic solution of sodium methoxide was added dropwise during 5minute. The reaction mixture was boiled for a half an hour andevaporated. The residue was stirred with 210 ml of dichloromethane for ahalf an hour, sodium chloride was filtered off, and the filtrate waswashed with 50 ml of water, then with 50 ml of saturated sodium chloridesolution, dried over magnesium sulfate and evaporated in reducedpressure. The crude product (9.8 g) was purified by chromatography toyield the title compound as a pale-yellow oil.

Yield: 2.9 g (29%)

Elementary analyses for C₁₅H₂₃N₃O₃

calcd. found C % 61.4 61.2 H % 79.0 79.1 N % 14.3 14.5

Example 67Diethyl-N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-iminocarbonateProcedure:

A mixture of 0.87 g (5 mmole) of1-aminooxy-2-hydroxy-3-(1-piperidinyl)-propane and 1.1 g (5.5 mmole) oftetraethyl orthocarbonate was stirred at 100° C. for 3 hours in thepresence of catalytic amount of p-toluenesulfonic acid. Afterevaporation the residue was purified by column chromotography (MerckKieselgel 60; eluent: chloroform/methanol-cc. NH₄OH 30:5:0.2) to givethe title compound as a pale-yellow oil.

Yield: 27.7% (oil)

¹³C-NMR (d, CDCl₃): 154.9 (s, C═N), 76.5 (t, N—OCH₂), 66.6 (d, CHOH),64.5 (t, CH₃ CH₂), 64.1 (t, CH₃ CH₂), 61:5 (t, CHCH₂N), 54.8 (t,piperidine), 26.0 (t, piperidine), 24.2 (t, piperidine), 14.9 (q, CH₃),14.1 (q, CH₃)

Example 68N-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-O-ethyl-N′-phenyl-isoureaProcedure:

18.4 g (0.1 mole) of ethyl N-phenyl-chloroformimidate (F. Lengfeld andJ. Stieglitz: Am. Chem. J. 16 70 (1894)) and 16.2 g (0.1 mole) of1-aminooxy-2-hydroxy-3-[(1,1-dimethylethyl)amino]-propane [Ger.Off. 2651 083] were dissolved in 200 ml of tetrahydrofurane, 13.9 ml (0.1mole) of triethylamine was added and the mixture was stirred at roomtemperature for 10 hours. Triethylamine hydrochloride that formed wasfiltered, and the filtrate was evaporated in vacuum, the residue wasdissolved in 200 ml of chloroform and washed with 50 ml of water. Theorganic layer was dried over sodium sulfate, filtered and evaporatedunder reduced pressure. The crude oily residue was purified bychromotography to give the title compound as a pale yellow oil.

Yield: 18.5 g (59.8%)

Elementary analysis for C₁₆H₂₇N₃O₃

calcd. found C % 62.1 62.3 H % 8.8 8.5 N % 13.6 13.7

Example 69 N-[3-(1-piperidinyl)-propoxy]-O-phenyl isocarboxamideProcedure:

3.16 g (20 mmole) of 1-aminooxy-3-(1-piperidinyl)-propane were dissolvedin 50 ml of benzene, 2.4 g (20 mmole) of phenyl cyanate was added andthe mixture was stirred at room temperature for 12 hours. Further 0.16 g(1.3 mmole) cyanate was added and the mixture was stirred further 12hours. After evaporation the residue was dissolved in methanol and thesolution was clarified by activated carbon and evaporated. The productwas crystallized from ethyl acatate/ethyl alcohol to give whitematerial.

Yield: 46.9%

Mp.: 63-70° C. (ethyl acetate)

¹³C-NMR (d, D₂O): 152.4; 129.9; 125.8; 119.9; 70.3; 57.4; 54.3; 53.2;23.0; 22.7; 21.0.

Example 70N-[2-Hydroxy-3-(1-piperidinyl)propoxy]-N′-pentamethylene-O-ethyl-isoureaProcedure:

2.7 g (0.01 mole) ofdiethyl-N-[2-hydroxy-3-(1-piperidinyl)propoxy]-iminocarbonate (see inExample 67) and 0.99 ml (0.01 mole) of piperidine were dissolved in 40ml of tetrahydrofurane and stirred at room temperature for 2 hours, thenevaporated to dryness. The residue was purified by chromatography toyield the title compound as an oil.

Yield: 2.1 g (67.1%)

Elementary analysis for C₁₆H₃₁N₃O₃

calcd. found C % 61.3 61.1 H % 10.0 9.8 N % 13.4 13.6

Example 71N,N-dimethyl-N′-[2-hydroxy-3-(1-piperidinyl)-propoxy]-N″-phenyl-guanidinehydrochloride Procedure:

1150 mg (6.58 mmole) of 1-aminooxy-2-hydroxy-3-(1-piperidinyl)-propane(Ger. Off. 2 651 083) was dissolved in chloroform and 750 mg of Na₂CO₃was added, then a solution of 1206 mg (6.58 mmole) ofN,N-dimethyl-N′-phenyl-chloroformamidine (BR 888646/1959, Bayer, auth.:Kühle and Eue L.; CA 57, 136961/1962/) in 10 ml of chloroform was addeddropwise. After 5 hours the solid state was filtered and the filtratewas evaporated. This residue (1800 mg oil) was dissolved in 10 ml ofethyl acetate, and the product was precipitated by addition of 10.46 mlof 0.54 N HCl/ethyl acetate. The yellow precipitate was filtered off,washed, and finally recrystallized from acetone, after ethyl acetate.

Yield: 28%

Mp.: 127 129° C.

IR (KBr): 3220, 2093, 2840, 2690, 2620, 1608, 1580, 1475, 1433, 1375,1250, 1070, 1050, 1000, 925, 900, 760, 705 cm⁻¹

Example 72N-[3-[1,1-dimethylethyl)amino]-2-hydroxypropoxy]-N′-phenyl-guanidineProcedure:

3.1 g (0.01 mole) ofN-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-O-ethyl-N′-phenyl-isourea(see in Example 68) was dissolved in 20 ml of tetrahydrofuran and 200 mlof 25% of ammonium-hydroxide solution and 0.26 g (5 mmol) of ammoniumchloride were added and the mixture was kept at room temperature for 15hours. The mixture was evaporated to dryness and purified bychromatography to yield the title compound as an oil.

Yield: 1.7 g (60.7%)

Elementary analysis for C₁₄H₂₄N₄O₂

calcd. found C % 60.0 60.2 H % 8.6 8.9 N % 20.0 19.8

Example 73N,N′-diphenyl-N-[2-hydroxy-3-(1-piperidinyl)-propoxy-benzenecarboxamidinehydrochloride Procedure:

3.55 g (20 mmol) of 3-piperidino-2-hydroxy-1-propane was dissolved in2.5 ml of water, 0.8 g (20 mmol) of NaOH was added, and the mixture wasstirred at room temperature for 1 hour. Then a solution of 6.49 g (20mmol) of N,N′-diphenyl-N-hydroxy-benzenecarboxamidine hydrochloride in60 ml of ethyl alcohol was added dropwise, and further 0.8 g (20 mmole)NaOH. The obtained yellow suspension was boiled for 2 hours. Then theprecipitated sodium chloride was filtered off, washed with ethylalcohol. Solvent was evaporated and 40 ml of ethyl acetate was added andtwo times was extracted with 40 ml portions of distilled water. Theorganic phase was dried over sodium sulfate, filtered. The product wasprecipitated by addition of 5.5 ml of 3.67 N HCl/ethyl acetate. Theprecipitate was filtered off, washed, and finally recrystallized frommethanol/ether.

Yield: 42%

Mp.: 151-155° C. (methanol/ether)

¹³C-NMR (d, CDCl₃): 159.6, 148.0, 140.9, 131.0, 129.7, 129.3, 128.9,128.5, 127.9, 127.5, 64.2, 60.2, 54.5, 22.7, 21.9.

Example 74N-[3-(1-piperidinyl-propoxy]-N-methyl-N′-phenyl-O-ethyl-isocarboxamide

Procedure for the preparation of this compound is the same as written inthe Example 68, using1-methylaminooxy-2-hydroxy-3-(1-piperidinyl)-propane andethyl-N-phenyl-chloroformimidate as starting materials.

Yield: 56% (oil)

Elementary analysis for C₁₇H₂₉N₃O₃

calcd. found C % 66.4 66.2 H % 9.5 8.9 N % 13.7 13.9

Example 75N-[2-hydroxy-3-(1-piperidinyl)propoxy]-N-methyl-N′-phenyl-guanidine

Procedure for the preparation of this compound is the same as written inthe Example 72, usingN-[2-hydroxy-3-(1-piperidinyl)propoxy]-N-methyl-N′-phenyl-β-ethyl-isourea(see Example 74) as starting materials.

Yield: 43% (oil)

Elementary analysis for C₁₆H₂₆N₄O₂

calcd. found C % 62.7 62.2 H % 8.5 8.8 N % 18.3 18.4

Example 76N,N,N′-trimethyl-N′-[3-(1-piperidinyl)-propoxy]N″-phenyl-guanidineProcedure:

344 mg (2.0 mmole) 1-methylaminooxy-2-hydroxy-3-(1-piperidinyl)-propanewas dissolved in chloroform and 220 mg of Na₂CO₃ was added, then asolution of 438 mg (2.0 mmole) ofN,N-dimethyl-N′-phenyl-chloroformamidine hydrochloride in 3 ml ofchloroform was added dropwise. After 8 hours the solid state wasfiltered and the filtrate was evaporated. This residue was dissolved inethyl acetate and the product was extracted by addition of HCl solution(pH=1) to water. The aqueous phase was made alkaline then by addition of2N NaOH solution to pH=11, and extracted with ethyl acetate. The organicphase was evaporated, and the further purification was made by columnchromatography to give the title compound as a yellow oil.

Yield: 10% (oil)

¹³C-NMR (d, CDCl₃): 156.6, 150.5, 128.4, 120.5, 70.0, 55.9, 54.4, 40.6,39.4, 25.8, 25.6, 24.3.

Example 77/R/(+)-N-[2-hydroxyl-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride (Z)-2-butenedioate) Procedure:

2.16 g (3.26 mmole) of(S)—N-[2-[2-(R)-(1,1-dimethylethyloxycarbonylamino)-3-phenylpropionyloxy]-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride (Z)-2-butenedioate (1:1) (see Example 18) was suspended in 40ml of methanol and boiled for 1 hour, then evaporated to dryness. Theresidue was triturated with 20 ml of ethyl acetate, the precipitate wasfiltered, washed with ethyl acetate. This crude product wasrecrystallized in isopropyl alcohol to yield the title compound:

Yield: 1.16 g (85%)

Mp.: 136-137° C.

[∀}_(D:+)5.6° (c=1, MeOH, t=27° C.)

Example 78 N-(3-piperidino-1-propoxy)-3-pyridinecarboximidoyl chloridedihydrochloride

After cooling to 0° C. a mixture of 10 ml of distilled water and 4.36 mlof concentrated hydrochloric acid, 2 g (7.62 mmoles) ofN-(3-piperidino-1-propoxy)-3-pyridinecarboxamidine (see Example 31) areadded under stirring. To the yellow solution 2.7 g (3.81 mmoles) ofsodium nitrite dissolved in 10 ml of water are added dropwise at −5° C.during 30 minutes. After stirring the greenish solution at −5° C. for1.5 hours, the pH of the solution is adjusted to 10 by adding 1 Naqueous sodium hydroxide solution under cooling, then the solution isextracted 3 times with 40 ml of chloroform. The organic phase is washedwith 20 ml of water, dried over sodium sulfate and evaporated. Theresidue is purified by column chromatography (Merck Kieselgel 60;eluent:chloroform/methanol 1:1) to obtain 1.7 g (79.2%) of the basecorresponding to the title compound.

The title hydrochloride is prepared from the base obtained by adding anethanolic solution of hydrogen chloride, m.p.: 165-167° C.

IR (KBr): 3015, 2945, 2515, 2088, 1982, 1600, 1570, 1437, 1402, 1200,1060, 988, 912, 808 cm⁻¹.

The above starting material can be prepared as follows:

After dissolving 2.86 g (51.06 mmoles) of potassium hydroxide in 20 mlof abs. ethanol, 6.45 g (47.0 mmoles) of 3-pyridinecarboxamide oxime areportionwise added while stirring. After dissolution, 7.7 g (47.66mmoles) of 1-(3-chloropropyl)piperidine dissolved in 5 ml of ethanol aredropwise added. After 9-hour reaction, the precipitated potassiumchloride is filtered off, the ethanolic solution is clarified byactivated carbon and evaporated. After taking up in 100 ml ofchloroform, the evaporation residue is washed 3 times with 100 ml of 1 Nsodium hydroxide solution each, then with 50 ml of water. Afterseparation, the organic phase is dried over sodium sulfate, filtered andevaporated. The oily residue becomes crystalline on cooling. Thecrystals are triturated with about 20 ml of ether, filtered and dried togive a beige product in a yield of 4:8 g (38.9%).

IR (KBr): 3422, 3107, 2937, 2870, 2819, 1640, 1479, 1391, 1309, 1194,1123, 1059, 1042, 982, 916 cm⁻¹.

Following the process described in the previous example the followingcompound was prepared:

Example 79 O-(3-piperidinopropyl)-3-nitro-benzhydroximyl chloridehydrochloride

Yield: 50%

Mp.: 173-175° C.

IR (KBr): 3420, 2926, 2953, 2649, 2546, 1514, 1591, 1533, 1452, 1354,1259, 1252, 1049, 994, 733 cm⁻¹.

FORMULATION EXAMPLES Example 1 Tablet

Tablet containing 50 mg active material is prepared from the followingcomponents:

N-[2-hydroxy-3-(1-piperidinyl)-propoxy}-2-thiophene-carboximidoyl

chloride monochloride 50.0 mg corn starch 100.0 mg  lactose 95.0 mg talc 4.5 mg magnesium stearate  0.5 mg

The active compound is finely ground, mixed with the additives, themixture is homogenized and granulated. The granulates are pressed intotablets.

Example 2 Tablet

Tablet containing 5 mg active material is prepared from the followingcomponents:

N-[2-hydroxy-3 (1-piperidinyl)-propoxy]-2-nitro-benzimidoyl

chloride monochloride 50.0 mg  corn starch 75.0 mg  lactose 7.5 mgcolloidal silica 7.5 mg magnesium stearate 5.0 mg

The composition is prepared from the above components in accordance withexample 1.

Example 3 Tablet

Tablet containing 5 mg active material is prepared from the followingcomponents:

N-[2-benzyloxy-3-(1-piperidinyl)-propoxy]-3-pyridine-carboximidamide-

(Z)-2-butenedioate (1:1) 5.0 mg corn starch 75.0 mg  gelatin 7.5 mgmicrocrystalline cellulose (Apical) 25.05 mg  magnesium stearate 2.5 mg

The composition is prepared from the above components in accordance withexample 1.

Example 4 Capsule

Capsule containing 10 mg active material is prepared from the followingcomponents:

N-[2-palmitoyloxy-3-(piperidinyl)-propoxy]-3-pyridine-

carboximidamide monohydrochloride 10 mg lactose 80 mg corn starch 25 mgtalc  3 mg colloidal silica  3 mg magnesium stearate  2 mg

The active material is mixed with the additives, the mixture ishomogenized and filled into gelatine capsules.

Example 5 Capsule

Capsule containing 20 mg active material is prepared from the followingcomponents:

N-[2-hydroxy-3-(piperidinyl)-propoxy]-2-thiophene-

carboximidoyl chloride 20 mg monohydrochloride microcrystallinecellulose (Apical) 99 mg amorphous silica  1 mg

The active material is mixed with the additives, the mixture ishomogenized and filled into gelatine capsules.

Example 6 Dragée

Dragée containing 25 mg active material is prepared from the followingcomponents:

N-[3-[(1,1-dimethyl-ethyl)-amino]-2-hydroxy-propoxy)-3-

trifluoromethyl-benzamidine hydrochloride 25 mg carboxymethyl cellulose295 mg  stearic acid 20 mg cellulose acetate phtalate 40 mg

The active material is mixed with the carboxymethyl cellulose andstearic acid, and the mixture is granulated in the solution of celluloseacetate phtalate in 200 ml ethanol-ethyl acetate. A core is pressed fromthe granulate which is covered by aqueous polyvinylpirrolidone solutioncontaining 5% sugar.

Example 7 Injection

Injection solution is prepared from the following components:

N-[2-hydroxy-3-piperidinyl)-propoxy]-2-nitro-

benzimidoyl chloride 5.0 g sterile physiological saline solution 2.0 ml

Example 8 Ointment

Ointment is prepared from the following components:

N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-2-thiophene

carboximidoyl chloride 7.5 g monohydrochloride stearic acid 18.0 g cetylstearyl alcohol 15.0 g glycerin monostearate 4.0 g sodium lauryl sulfate1.5 g methyl p-hydroxy benzoate 0.2 g distilled water 150 ml

The stearic acid, cetyl stearyl alcohol and glycerine monostearate aremelted together. The sodium lauryl sulfate and methyl-p-hydroxy benzoateare dissolved in 100 ml water under slight warming and then added to thelipophylic components while stirring until the temperature decreases toroom temperature. Subsequently, the solution of active compound in 50water is added and thoroughly mixed.

Example 9 Ointment

Ointment is prepared from the following components:

N-[2-hydroxy-3-piperidinyl-propoxy]-2-nitro-

benzimidoyl chloride monohydrochloride 7.0 g polysorbate 4.0 g liquidparaffin 4.0 g cetyl stearyl alcohol 12.0 g white vaseline 20.0 gglycerin monostearate 4.0 g methyl p-hydroxy benzoate 0.2 g ethylalcohol 1.8 g distilled water 150 ml

The composition is prepared as described in Example 8.

Example 10 Cream

Cream is prepared from the following components:

N-[2-hydroxy-3-piperidinyl-propoxy]-4-pyridine-carboximidoyl

chloride (Z)-2-butenedioate (1:) 10.0 g white vaseline 90.0 g white wax3.0 g cetyl stearyl alcohol 3.0 g sodium tetraborate 4.0 g methylp-hydroxy benzoate 0.2 g distilled water 90 ml

The solution of the water-soluble components is added to the warmmixture of the lipophylic components as in Example 8, and the aqueoussolution of the active compound is added to the final emulsion.

Effect of N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride maleate on Cellular Expression of HSP Examined on TranslationalLevel 1. Background

Experiments set forth in this section were conducted to determinewhether N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride maleate acts to increase the expression of molecular chaperonby a cell. The accumulation of different heat shock proteins subsequentto a period of exposure to heat shock alone, and to heat shock incombination withN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate administration, was examined by adding 10⁻⁵ M ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate before, during, or immediately after hyperthermic treatment ofheart myogenic cells (H9c2 cells).

2. Materials & Methods 2.1 Cell Culture Conditions

The embryonic rat heart-derived cell line H9c2 was obtained fromEuropean Collection of Animal cell Cultures (ECACC) (88092904). Thecells were maintained at 37° C. in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% fetal calf serum (GIBCO) in JOUAN C02thermostat (5% CO₂).

2.2 N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride maleate Treatment and Heat Shock Conditions

Heat shock was performed at 43° C. in CO₂ thermostat for the given timeintervals (20, 40, 60, 90 and 120 min.). Cells were then taken back to37° C. for 6 hours and proteins were extracted for SDS-PAGE. WhenN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate was added before heat shock. 10⁻⁵ MN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate was administered for 16 hours before the stress. In other set ofexperiments,N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate was added in 10⁻⁵ M concentration right after heat stress,during the 6 hours recovery period. The experiments were repeated threetimes.

2.3 Western Blot

For SDS-P AGE, cells were grown in 6 cm Petri dishes. The amount of thecells at the start of the experiment was 8×10⁵ and were stillsubconfluent when proteins were extracted. After the 6 hours recoverycells were washed two times in PBS then scrapped from the surface of thedishes in PBS. Then cells were spun for 5 min. at 1500 rpm and taken upin 100:1 modified solubilizing buffer (Molecular Cloning, A LaboratoryManual, Ed. Sambrook. Fritsche. Maniatis, Bold Spring Harbor LaboratoryPress (1989)) containing 50 mM Tris-HCl, pH8.0; 5 mM EDTA; 150 mM NaCl;15 Tritox N-100; 1 PMSF; 2:g/ml aprotinin; 1:g/ml chymostatin; 1:g/mlpepstatin; and sonicated for 3×2o sec (2 min. intervals, setting 8).

Protein concentration was determined from 5:1 samples by the Bradfordassay (M. M. Braford, Anal. Biochem., 72: 248-254 (1976)) in threeparallel. Samples were adjusted to 100:g/30:1 with the above buffer andthe next buffer so that the concentration of the buffer in the samplewill be: 110 mM Tris-HCl pH 6.8, 8.3 mM mercaptoethanol, 3% SDS, 3%glycerol and some bromophenol blue and shaken at room temperature for 30min.

Electrophoresis was carried out according to Laemmli (U. K. Laemmli,Nature, 227:680-685 (1970)) on two 8-18% polyacrylamide gel at constantvoltage 50 V for overnight. Proteins were either stained with CoomassieBrilliant Blue R-250 or transferred to Immobilone PVDF membrane(Millipore) at constant current (300 mA) for 3 hours at 4° C. intransfer buffer (10 mM CAPS, pH 11, 10% methanol) (Protein BlottingProtocols! for the Immobilon-P Transfer Membrane, 3. Laboratory Manual,Millipore). After transfer, non-specific sites of the membrane wereblocked with 2% BSA in TPBS (phosphate buffered saline with 0.1% Tween82) for overnight at 4° C. The blot was incubated with GRP94 monoclonalantibody (SPA-850, StressGen) diluted 1:3000, with HSP60 monoclonalantibody (SPA-600, StressGen) with 1:2700 dilution, with HSP72monoclonal antibody (C92F34-5, StressGen) diluted 1:1250 or with HSP90monoclonal antibody (AC88, StressGen) diluted 1:2000, for 1 hour at roomtemperature. Then it was washed with TPBS buffer for one hour. andincubated with horseradish peroxidase conjugated anti-rat (Sigma, 1:4000dilution, for GRP-94) or anti-mouse (Sigma, absorbed with human and ratserum proteins, 1:3000 dilution, for Hsp60, HSP72 or HSP90) secondaryantibody for 1 hour respectively. After successive washing with TPBS themembrane was developed with ECL system (Amersham).

A total-protein dilution series was blotted and developed parallel withthe samples every time, and a calibration curve was calculated. Thechanges in the stress protein content was quantified using a Bio-Raddensitometer (Model 1650) and a Hewlett-Packard Integrator (HP 3394A)and corrected according to the calibration curve. When making thecalculations the densitometric data of the given protein band at 37° C.without N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride maleate treatment was considered as 100% and all the otherintensities were compared to that sample.

2.4 Statistical Analysis

Data are reported as means±SE. Statistical comparison and calculationwas performed by one-way analysis of variance with Posthoc Newman-Keulstest (Pharmacological Calculation System). Statistical significance wasdefined as P<0.05.

3. Results

Hsp60: N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride maleate treatment accomplished at 37° C. has no measurableeffect on the level of this hsp. Heat shock at 43° C. alone, lasting for20 min. can increase the amount of hsp60 almost by twofold. By extendingthe duration of heat treatment, no further elevation could be observedin the level of this protein. WhenN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate was added at a concentration of 10⁻⁵ M 16 hours before heatstress, the amount of hsp60 increased by about fivefold (compared to 37°C. control) even if samples exposed to 20 min. heat treatment. Thiseffect of N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride maleate on the level of hsp60 was evident also in samples heattreated for 40 and 60 min., respectively, however started to declinethereafter.N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate added during the recovery phase was also effective though to asignificantly lesser extent then observed by adding this compound beforethe stress.

Hsp72: The amount of resting hsp72 was rather low in H9c2 cells, theeffect of different treatments on hsp70 level, however, was dramatic.There was already a significant increase at 20 min. heat shock, and at40 min. heat treatment the amount was almost 10 times higher than thatof detected in control cells. Heat treatment at longer duration resultedin no significant change compared to 40 min. samples. Administration ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate before heat stress had a very profound effect. At 60 min. heatstress the hsp72 level increased by about 50 times when comparing to 37°C. samples, but a significant increase could be detected already at heatstress lasting for 40 min. These highly induced amounts of hsp72 werestable in cells heat shocked for 120 min.N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate added during the recovery phase was also effective though to alesser degree.

Hsp90: At 20 min. heat treatment, high temperature shock alone wasunable to induce elevated level of HSP90, however, ifN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate added before the heat stress, the level of Hsp90 increased byabout fivefold. The highest effect of the drug preincubation could beobserved following its combination with 60 min. heat treatment. It wasinteresting to note, that in the case of HSP90N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate added following 43° C. 60 min. treatment was as effective (ifnot more) as if added before high temperature stress. Obviously,incubation at high temperature longer than 90 min. the effect ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate is fading.

Grp94: The formation of the stress protein Grp94 was induced following a20 min. heat shock. This effect peaked at thermal treatment for 60 min.whereas a sharp decline has already been detected in case of 90 min.samples. The capability ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]3-pyridinecarboximidoyl chloridemaleate to induce the level of Grp94 was the most pronounced in case ifthe compound was added before the stress but significant raise could beseen if the preaddition of the drug was combined with a 20 min. longheat shock (where there was a 4 times increase compared to 43° C.treatment). On the other hand, addition of the compound during recoveryfrom heat stress lasting for 40 to 60 min. was almost effective as theadministration ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate for 16 hours long before the stress.

FIG. 2 shows the Western blot analysis of proteins from H9c2 cells.Probes used are: hsp60 antibody, shown in (1); hsp72 antibody, shown in(2); hsp90 antibody, shown in (3) and grp94 antibody, shown in (4). Lane(−) represents cells kept in the absence ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate at 37° C.; and lane (+) represents cells kept in its presence(concentration of 10⁻⁵ M for 16 hours). Heat shock at 43° C. wasperformed for the times indicated on the figure.N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate at 10⁻⁵ M was added 16 hours before the heat treatment (lane(B)) or during the recovery period (lane (A)).

An overview of the effect of the various treatments on the amount ofdifferent hsp in H9c2 heart cells is provided in FIG. 1. The amount ofstress proteins in cells exposed to heat stress (43° C.) alone isrepresented by (A); the amount of stress proteins in cells treated with10⁻⁵ M N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride maleate before the heat treatment is represented by (B); andthe amount of stress proteins in cells treated with 10⁻⁵ MN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate during the six hour recovery period is represented by ©. Thehorizontal axis represents the time duration of the heat treatment andthe vertical axis represents the relative amount of stress proteins.

Heat treatment alone induced all kinds of HSPs investigated in thisstudy. The increase was the less pronounced in the case of hsp60. Thelevel of hsp60 elevated up to about twofold after exposure of cells to20 min. heat, whereas no further increase could be detected at longerheat treatments. The largest effect of heat stress could be seen forhsp72, as its amount increased to about 12 times. WhenN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate was added 16 hours before the heat treatment the level of allHSPs increased further up to at least twofold as compared to thatobserved upon heat stress. It was also clear that the level of hsp72increased in a much higher extent amongst HSPs by combining heat stressand N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride maleate if compared again to the induction detected for heatstress alone. WhenN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate was administered during the recovery phase almost in all casesexamined, content of hsp(s) increased, but again elevation of hsp72 wasthe most pronounced.

4. Discussion

Western blot analysis revealed a remarkable accumulation of differentclasses of HSPs examined after the exposure of heart cells to heatshock. Addition ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate either before or after high temperature stress multiplied theeffect of heat treatment upon the production of HSPs. Accordingly,N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate acts in synergy with temperature stress by inducing theformation of all classes of molecular chaperones.

Effect ofN-[2-hydroxy-3-(1-Piperidinyl)-propoxy]-3-Pyridinecarboximidoyl ChlorideMaleate on Cellular Expression of HSP Examined on TranscriptionalLevel 1. Background

Brief exposure to ischemia (e.g., by repeated stunning) can preconditionthe heart and protect it from subsequent lethal ischemia, as evidencedby decreased incidence of ventricular fibrillation, reduced infarct sizeand better recovery of regional myocardial function during thereperfusion of ischemic heart. Such precondition has been demonstratedto induce the expression of HSPs, especially hsp72. In this section, theeffect of N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3pyridinecarboximidoylchloride maleate on the expression of hsp72 is investigated by examiningthe mRNA accumulation in a cell following ischemia and comparing it tothe situation where ischemia is combined with administration ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate.

2. Materials & Methods 2.1 Induction of Heat Stress

Experiments were carried out in SPRD male rats. Animals wereanaesthetized with Nembutal in a dose of 60 mg/kg/i.p. Body temperatureof the rats were maintained with an infra lamp placed over the abdomenand the rectal temperature was measured. After a 25-40 minute period thetemperature of the rats increased to 42.0-42.2° C. and this temperaturewas maintained for 15 minutes. After a recovery period (2 hrs), tissuesamples were collected from the left and right ventricles.

2.2 Induction of Cardiac Ischemia

Experiments were carried out in SPRD male rats. The animals wereanaesthetized with Nembutal in a dose of 60 mg/kg/i.p. After opening thechest and pericardium, the LAD coronary artery was occluded for 5minutes and then the incidence and duration of ventricular tachycardiaand fibrillation in the reperfusion period (10 minutes) wereinvestigated. Tissue samples were collected from the left and rightventricles.

2.3 Northern Blot Method

Total RNA was extracted using RNAgents kit (Promega) according to themanufacturer's instructions (Protocols and Applications Guide, 2ndedition, 1991, Promega Corporation). Briefly, the frozen tissue samples(the tissue samples from the left and right ventricles of rats subjectedto heat stress or cardiac ischemia). The tissue samples weighing 50 to100 mg were homogenized in 1.0 ml denaturizing solution at +4° C. byBrinkman homogenization. Then 1/10 vol. 3M sodium acetate (pH 4.0) wasadded and the homogenate were extracted with acidic phenol(phenol:chloroform:isoamylalcohol 25:24:1) for 10 seconds by vortex. Thesample was incubated on ice for 15 minutes, and then centrifuged (4 C;20 min, 10,000×g). The aqueous phase was transferred to a newEppendorf-tube, the process was repeated and the aqueous wasprecipitated at −20° C. overnight. Following centrifugation (4° C.; 20min. 10,000×g) the precipitate was washed twice with 95% ethanol anddried at room temperature. The RNA was suspended in 20:1 DEPC-treatedwater. Eight:1 g of total RNA was run on formaldehyde-agarose gel bycapillary transfer, the RNA on the gel was blotted onto nylon membraneaccording to the manufacture's instructions (Zeta-Probe GT, BioRad).

The hsp72 mRNA content of individual samples was compared with the mRNAlevel of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene levelof the corresponding probes. DNA probes (full length human hsp70 cDNAand Apa-Ncol fragment of the rat GAPDH cDNA) was labeled with alpha-³²PCTP using Random Primed DNA Labeling Kit (USB). Radiolabeled DNAfragments were purified on Sephadex G-50 (Pharmacia) column as described(Ausubel et al. (eds)): Current Protocols in Molecular Biology: JOHNWILEY & SONS: 1987).

Prehybridizations were carried out at 65° C. in H-buffer (0.25M Na₂HPO₄,pH 7.2, 7% SDS) for 15 minutes. Hybridizations were carried outovernight (65 C; H-buffer) with isotope labeled probe concentration ofat least 10⁶ cpm/ml. The membrane was washed with 20 mM Na₂HPO₄, pH 7.2,5% SDS (65 C; 2×15 min.) and evaluated by autoradiography. The samemembrane was used for the hsp70 probe as well as for the measurement ofGAPDH and mRNA used as internal standard.

3. Results

Coronary occlusion for 5 minutes was followed by reperfusion whichprovoked ventricular tachycardia and fibrillation in the rats.N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate pretreatment (0.5-,0.75-, 1.0 mg/kg of body weight i.v. 5 min.before the occlusion) reduced significantly the mean duration ofventricular tachycardia and improved the survival rate by preventingventricular fibrillation.

Whereas all animals (n=6) from the control group died during the phaseof reperfusion, theN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate treated (100 mg/kg p.o.) ones not only survived the reperfusionafter the 5 min. occlusion, but a highly increased expression of hsp72gene was detected in their heart muscle preparations.

FIG. 3 is the Northern blot analysis of total RNA isolated from leftventricles of rat heart, illustrating the results obtained from theexperiment. Control (lane 1); heat treated (lane 2); sham operated (lane3); ischemia (lane 4);N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate plus ischemia (lane 5); andN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate (lane 6). GADPH was measured as an internal probe. For heatshock, the rectal temperature was maintained at 42° C. for 15 minutes.

It is noted that in the absence of stress, administration ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate alone was unable to activate the hsp72 gene.

Protective Effect of Hydroxylamine Derivatives of the Invention AgainstCardiac Ischaemia

Male Sprague-Dawley rats (380-450 g b.w.) were anaesthetized withsodiumpentobarbital (Nembutal 60 mg/kg body weight, i.p.) andartificially ventilated with room air (2 ml/100 g; 54 stroke/minutes)via tracheotomy. The right carotid artery was catheterized and connectedto a pressure transducer (BPR-01, Stoelting) for the measurement ofsystemic arterial blood pressure (BP) by means of a preamplifier (Hg-02,Experimetria). Hydroxylamine derivatives disclosed in Example 5 wereadministered via the venous cannule to jugular vein (i.v.) and orally(p.o.). Heart rate (HR) was measured by a cardiotachometer (HR-01,Experimetria). The electrocardiogram (ECG standard lead II) was recordedon a devices recorder (MR-12, Medicor) by means of subcutaneous steelneedles electrodes. The chest was opened by a left thoracotomy and theheart was exteriorized by a gentle pressure on the right side of the ribcage. A 4-0 silk suture was quickly placed under the main left coronaryartery as described by Selye et al. (1960). The heart was carefullyreplaced in the chest and the animal left to recover. Rectal temperaturewas monitored and kept constant at 37 C. The experimental protocol wasinitiated with a 15 minute stabilization period during which theobservation of a sustained blood pressure less than 70 mmHg and or theoccurrence of anhythmia led to exclusion. Myocardial ischemia wasinduced with coronary occlusion for 5 minutes and reperfusion allowedfor 10 minutes.

During the entire experiment BP, HR and ECG were continuously registeredon a multiscriptor (R61-6CH, Medicor). Hydroxylamine derivatives, thetautomeric forms of which are represented by structures (I) and (II),were administered 5 to 60 minutes before the occlusion after i.v. andp.o. treatment, respectively. The doses of the hydroxylamine derivativesof Example 5 were 0.5; 0.75; 1.0 mg/kg i.v. and 100 mg/kg of body weightp.o., while the reference substance Bepridil was given in a dose of 1.0mg/kg i.v.

The mean duration of ventricular tachycardia (VT) and/or ventricularfibrillation (VF) during the first 3 minutes of reperfusion was analyzedby one-way analysis of variance. The incidence of VF was analyzed usinga chi-square test. The haemodynamic variables were analyzed using achi-square test. The haemodynamic variables were analyzed usingStudent's “t”-test. The critical level of significance was set atp<0.05. All results were expressed as a means±S.E.M. Drugs wereadministered i.v. in a dose of 1 mg/kgbw 5 min. before the occlusion.

The hydroxylamine derivatives that are found to be particularlyadvantageous for protecting an animal against ischemic/reperfusioninjury are listed below. Survival (%) indicates the percent of animalssurvived the effects of 5 minutes coronary occlusion.

Code Survival (%) Example 77 i.v. 1 mg/kgbw 67 Example 78 i.v. 1 mg/kgbw100 Example 8 i.v. 1 mg/kgbw 100 Example 13 i.v. 1 mg/kgbw 60 Example 9i.v. 1 mg/kgbw 100 Example 10 i.v. 1 mg/kgbw 67 Example 7 i.v. 1 mg/kgbw80 Example 6 i.v. 1 mg/kgbw 100 Example 79 i.v. 1 mg/kgbw 100 Example 1i.v. 1 mg/kgbw 100 Example 16 i.v. 1 mg/kgbw 67 Example 65 i.v. 1mg/kgbw 78 Example 54 i.v. 1 mg/kgbw 80 Example 20 i.v. 1 mg/kgbw 100Example 22 i.v. 1 mg/kgbw 100 Example 47 i.v. 1 mg/kgbw 100 Example 39i.v. 1 mg/kgbw 60 Example 51 i.v. 1 mg/kgbw 75 Example 64 i.v. 1 mg/kgbw100 Example 56 i.v. 1 mg/kgbw 67 Example 57 i.v. 1 mg/kgbw 67 Example 58i.v. 1 mg/kgbw 100 Example 59 i.v. 1 mg/kgbw 86 Example 60 i.v. 1mg/kgbw 60 Example 61 i.v. 1 mg/kgbw 83 Example 55 i.v. 1 mg/kgbw 80Example 66 i.v. 1 mg/kgbw 57 Example 62 i.v. 1 mg/kgbw 57 Example 63i.v. 1 mg/kgbw 50 Example 4 i.v. 1 mg/kgbw 50 Control (non-treated) n =24 10

In addition to the above compounds, the following compounds have alsobeen found to provide advantageous results:

N-[2-hydroxy-3-(pyrrolidin-1-yl)-propoxy]-3-pyridincarboximidoylchloride (Z)-2-butendioate (1:1) (U.S. Pat. No. 5,328,906, Example 12, 1mg/kgbw i.v., survival %: 67):

N-[2-hydroxy-3-(diethyl-amino)-propoxy]-3-pyridincarboximidoyl chloridehydrochloride (1:1) (U.S. Pat. No. 5,328,906, Example 11, 1 mg/kgbwi.v., survival %: 62);

N-[2-hydroxy-3-prop-2-yl-amino)-propoxy]-3-pyridincarboximidoyl chloride(Z)-butendioate (1:1) (U.S. Pat. No. 5,328,906. Example 13/4, 1 mg/kgbwi.v., survival %: 60);

N-[2-hydroxy-3-(morpholin-1-yl)-propoxy]-3-pyridincarboxamidoyl chloride(Z)-2-butendioate (1:1) (U.S. Pat. No. 5,328,906, Example 12, 1 mg/kgbwi.v., survival %: 71);

N-[2-hydroxy-3-(1-piperidyl)-propoxy]-∀-(3,4-dimethoxy-phenyl)-acetimidoylchloride (U.S. Pat. No. 5,328,906, Example 14, 1 mg/kgbw i.v., survival%: 67);

N-[2-hydroxy-3-(tert-butyl-amino)-propoxy]-3-pyridincarboxamidoylchloride (Z)-2-butendioate (1:1) (U.S. Pat. No. 5,328,906, Example 13/5,1 mg/kgbw i, v., survival %: 57);

O-(3-piperidino-2-hydroxy-1-propyl)-benzhydroximic acid

chloride hydrochloride (U.S. Pat. No. 5,147,879, Example 1, 1 mg/kgbwi.v., survival 1%: 100);

N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboxamidoyl chloride(Z)-2-butendioate (1:1) (U.S. Pat. No. 5,147,879, Example 2, 1 mg/kgbwi.v., survival %: 100.20 mg/kgbw p.o. survival %: 100);

Effect of N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride maleate in Cell Membrane Repair and Preservation of MembraneFluidity

1. Alteration in Cell Membrane Fluidity Associated with Cellular InjuryInduced by Serum Deprivation Stress and Effect ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate in Reverting the Alternation in Fluidity

1.1 Background

One approach to modeling pathophysiological events caused by the stressof metabolic impairment accompanying diabetes is to decrease the levelof insulin in the culturing medium. Since insulin is provided by thesupplemented serum, partial or total deprivation seemed to be an optimaltool to detect changes in different regulatory levels of the cell.

Serum deprivation is widely used method for cell arrest in GUS phase,i.e. cell cycle synchronization (Ashihara. T. Methods of Enzymology,58:248-249 (1979)). It has been observed that cultured cells areundergoing apoptotic processes in the lack of serum supplementation(Cohen, et al., Adv. In Immunology 50:50-85 (1991)) and it has beenstudied as an alternative shock of staurosporin or toposiomeraseinhibitors in Balb/3T3 cells (Kulkarni. G. V, et al., J. Cell. Sci. 107:1169-1179 (1994)), or heat shock and glutamine deprivation in Ehrlichcells (Rowlands, A G. et al., Eur. J. Biochem. 175: 93-99 (1988)) toinvestigate the inhibited protein synthesis by the phosphorylation ofthe eukaryotic initiation factor (eIF2 alpha). Moreover, there areevidences on the induction of cytoprotection in serum deprived cells byadministering external HSP72 (Johnson, A. D. et al., In Vitro Cell Dev.Biol. Anim. 29A:807-812 (1993)). Increases in the relative synthesis ofHSP82 and HSP72 by serum deprivation (Toye, P. et al., Mol. Biochem.Parasitol. 35, 11-10 (1989)) was also shown.

Ischemic and hypoxic injury of the myocardium and other organs ismediated by progressive membrane dysfunction and damage. It was alsodemonstrated that alteration in membrane fluidity occurs duringmetabolic impairment of cardiac cells (Buja L. M. et al, In vivo 5:233-238 (1991)). Membrane fluidity primarily reflects the orientationand rate of the movement of membrane constituent lipids and anyalteration of its level greatly influences the fundamental membranefunction (Quinn, P. J. et al. Prog. Biophys. Molec. Biol. 53: 71-103(1989); Schlame, M. et al., Biochim. Biophys. Acta, 1045: 1-8 (1990)).

In this experiment, investigation was conducted to determine whetherchanges in plasma membrane fluidity participate in the development inthe cellular injury induced by serum deprivation and whether the serumdeprivation induced fluidity alteration can be reverted by theadministration ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate.

1.2 Materials and Methods

Experiments were carried out using WEH1 mouse fibrosarcoma and H9c2 ratheart muscle cell lines divided into three groups:

control (10% FCS fetal calf serum)

serum deprived

N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate (10⁻⁵ M) treated and serum deprived

1.2(i) Serum Deprivation and MTT Test

We have screened various cell lines, drug concentrations, pretreatmentand deprivation times and we have found the most appropriate conditionsas follows: 5×10⁴/ml H9c2 rat heart muscle cell (n=6) and WEHI mousefibrosarcoma cell (n=7) have been plated on 24 well plates in 10% FCSDMEM (Dulbecco modified Eagle's medium) and incubated for 2 hours at 37C, 5% CO₂. Medium has been discarded and replaced by 10% FCS DMEMcontaining 10⁻⁵ MN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate. After further incubation on the above circumstances for 6hours, plates have been washed intensively by PBS and serum deprived.Pretreated cells were administered withN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate until the end of the experiment. Following 18 hours starvation,most of the cells were detached, i.e., died, observed microscopically,in serum deprived culture, whileN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate pretreated cultures have shown similar picture to the controlcells. Cell viability was measured by MTT test based on the method ofPlumb et al. (Cancer Res. 49: 4435-4444 (1989)) The tetrazolium saltmethod involving conversion of MTT(3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) to coloredformazan by alive cells, served as an indirect measurement of cellviability. MTT has been added directly to the medium at a concentrationof 1 mg/ml. After 2 hr incubation at 37° C. in dark, the supernatant wasremoved and 200:1 of 0.05 M HCl in isopropanol was added immediately tothe cells. O.D. of the plates was read at 570 nm on an ELISA platereader (Labsystems Multiskan Biochromatic type:348). Experiments havebeen carried out in each case at least in triplicates. Relative cellviability was calculated defining control group as 100%.

1.2(ii) Determination of Steady-State Fluorescence Anisotropy

Cell suspensions of 1×10⁵ cells/ml in PBS were labeled by the additionof DPH-PA (3-[p-(6-Phenyl)-1,3,5-hexatrienyl]phenylpropionic acid)dissolved in tetrahydrofuran at a final concentration of 0.1:M andincubated for 10 min. at 37 C. The amount of organic solvent added was0.05% to avoid its effect on cell membranes. The membrane probe DPH-PAwas selected due to charge properties of the probe that enable DPH-PA tolocalize predominantly within the outer leaflet of the plasma membrane,with the diphenyl-hexatriene moiety intercalating between upper portionsof fatty acyl chains (Kitagawa, S., et al. J. Membrane Biol. 119:221-227(1991)). DPH-PA exhibits strong fluorescence enhancement upon binding tolipids, providing a means of evaluating fluorescence anisotropy as afunction of lipid ordering. Fluorescence measurements were carried outat 37° C. using a Quanta Master QM-1 T-format luminescence spectrometer(Photon Technology Int. Inc., NJ. USA) equipped with polarizers in theexcitation and in the two emission light path. Excitation and emissionwavelengths were 360 nm (5 nm slit width) and 430 nm (5 nm slit width),respectively. The measured fluorescence intensities were corrected forbackground fluorescence and light scattering from the unlabeled sample.The fluorescence anisotropy was calculated as

rs=(IVV−G.IVH)/(IVV+(2×Gx IVH))

where IVV and IVH are observed intensities measured with polarizersparallel and perpendicular to the vertically polarized exciting beam,respectively. The factor G equals IVH/IHH and corrects for the inabilityof the instrument to transmit differently polarized light equally andfor the difference in sensitivity of the two emission channel (Kitagawa,S., et al. J Membrane Biol. 119:121-227 (1991)). IHV and IHH are thefluorescence intensities determined at vertical and horizontal positionsof the emission polarizer when the excitation polarizer is sethorizontally.

1.2(iii) Statistical Analysis

Data are reported as means±SEM. Statistical comparison and calculationwas performed by one way analysis or variance with Posthoc Newman-Keulstest (Pharmacological Calculation System). Statistical significance wasdefined as P<0.05.

1.3 Results

1.3(i) Serum Deprivation as a Model to Test Cytoprotective Effect ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate

Serum deprived WEH1 and H9c2 cells showed 74.8% and 50.5% relative cellviability, respectively. In case if 10⁻⁵ MN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate was present in the culture medium the administration resulted inan almost total survival of WEHI (93%) and a high protection in H9c2heart muscle cells (82.75%). Decrease of relative cell viability byserum deprivation and its protection byN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate in both cell lines were significant.

1.3(ii) Effect of N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloride maleate on the Fluidity ofSerum Deprived Mammalian Cells

To examine the effect of serum deprivation on the plasma membranefluidity of cultured mammalian cells fluorescence anisotropymeasurements have been accomplished by using the plasma membrane probeDPH-PA. The physical properties of the cell plasma membranes weresignificantly altered by serum deprivation. Serum deprivation caused apronounced decrease in fluorescence anisotropy of DPH-PA that is anabnormal increase in plasma membrane fluidity in both cell modelsinvestigated. Upon the addition ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate we observed an almost complete preservation of the membranephysical state. These changes were obviously consistent with thetendencies described on cell viability.

1.4 Discussion

By resulting in metabolic impairments, deprivation of normal culturemedium in both type of cells studied reduced their viability as testedwith the mu method. This effect was almost fully reverted upon theaddition ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate. Deprivation of serum induced also prominent alterations in thefluidity of plasma membranes which is known to contribute to themembrane disfunction accompanying myocardial injury. In contrary, serumdeprived cells, grown in the presence ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate, were able to preserve (or retain) partially their normal plasmamembrane physical state.

Effect of Insulin andN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridinecarboximidoyl chloridemaleate on the Level of GRP-94 in STZ Diabetic Rat Liver 1. Background

Anoxia, glucose starvation and several other conditions that adverselyaffect the function of endoplasmic reticulum (ER) induce the synthesisof the glucose regulated class of stress proteins (GRPs) (Lin, H. Y., etal. Mol. Biol. Cell. 4:1109-1119 (1993)). The 94 kDa member of GRPs,GRP-94, 50% homologous to 90 kDa stress protein, is a lumenalcalcium-binding protein of ER. Together with other proteins of ER GRP-94appears to function as a molecular chaperon (Nigem, S. K., et al. J.Biol. Chem. 263; 1744-1749 (1994)). It is assumed that the accumulationof molecular chaperone GRP-94 should have a beneficial effect on therepairing of cellular damage induced by STZ diabetes in rats.Accordingly, in the experiments set forth herein we compared the variouslevels of GRP-94 in livers derived from healthy, diabetic,N-[2-hydroxy-3-O-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate treated and insulin plus N-[2hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate treated rats.

2. Materials & Methods

2.1 Test substances:N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate (BIOREX Ltd.) insulin (Protophane HM ini.)

2.2 Animals: Crl (VAF Plus) Wistar Male Rats (250-300 g)

Animals were housed 7 per cage at 23-25° C. at 50-60% relative humiditywith 12/12 hours light-dark cycle. Free access was given to chow and tapwater

2.3 Induction of Diabetes: A Single Dose of STZ (45 Mg/Kg i.v.) wasGiven in Fasting State.

2.4 Animals were Divided into the Following Groups:a. Healthy Animals1. Saline treated healthy for 1 weeks (n=5)2. Saline treated healthy for 2 weeks (n=5)3. Saline treated healthy for 4 weeks (n=5)4. N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride maleate treated for 1 weeks (n=7)5. N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride maleate treated for 2 weeks (n=7)6. N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride maleate treated for 4 weeks (n=7)b. STZ-Diabetic Animals7. Saline treated diabetic for 1 weeks (n=5)8. Saline treated diabetic for 2 weeks (n=5)9. Saline treated diabetic for 4 weeks (n=5)10. N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride maleate treated diabetic for 1 weeks (n=7)11. N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride maleate treated diabetic for 2 weeks (n=7)12. N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride maleate treated diabetic for 4 weeks (n=7)c. Insulinized STZ-Diabetic Animals13. 1-week insulin treated diabetic (n=7)14. 2-week insulin treated diabetic (n=7)15. 4-week insulin treated diabetic (n=7)d. N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride maleate Treated and Insulinized Diabetic Animals16. 1-week insulin andN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate treated diabetic (n=7)17. 2-week insulin andN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate treated diabetic (n=7)18. 4-week insulin andN-[2-hydroxy-3-(-1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate treated diabetic (n=7)

After the treatments liver was removed, and was immediately frozen at−70° C. in liquid nitrogen.N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate treatment (wherever applicable): 20 mg/kg/day, p.o. Insulintreatment: twice daily in a dose required to maintain normal glucoselevel.

2.5 Determination the Level of GRP-94.

Extraction of Total Soluble Protein from Rat Liver.

All steps were carried out at 0-4 C. Rat livers (about 15-20 g) werehomogenized with a domestic mixer for 2 min. in 80 ml of a modifiedsingle detergent lysis buffer solution containing 50 mM Tris-HCl pH 8.0,5 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100 and 1-1 mM pro-teaseinhibitors (PMSF, benzamidine, amino-caproic-acid). The homogenate wascentrifuged at 20000×g for 30 min. in a Servile RC 28S centrifuge. Themajority of the supernatant was frozen to −20° C. as a stock sample), 1ml was used for the analysis.

Protein concentration was determined by the Bradford assay (Guide toProtein Purification. Methods in Enzimology. vol. 182. M. P. Deutscher(Ed.), Academic Press (1990)) in three parallel and was adjusted to 5mg/ml.

Electrophoresis and Immunoblotting

Laboratory techniques for electrophoresis and immunoblotting aredescribed in detail in Molecular Cloning, A Laboratory Manual, Ed.Sambrook, Fritsche, Maniatis, Bold Spring Harbor Laboratory Press(1989): Protein Blotting Protocols for the Immobilon-P TransferMembrane, 3. Laboratory Manual, Millipore: and U. K. Laemmli, Nature:227: 680-685 (1970). Each sample consisted of 1.8 mg protein wassolubilized for gel-electrophoresis with 0.6 ml buffer containing 110 mMTris-HCl pH 6.8, 8.3 mM mercaptoethanol, 3% SDS, 3% glycerol and somebromophenol blue and shaken at room temperature for 30 min.Electrophoresis was carried out on 8% polyacrylamide gel with 30 mgprotein per lane at constant voltage 50 V for overnight. Proteins wereeither stained with Coomassie Brilliant Blue R-250 or transferred toImmobilone PVDF membrane (Millipore) at constant current (300 mA) for 3hours at 4° C. in transfer buffer (10 mM CAPS pH 11.10% methanol).Non-specific sites of the membrane were blocked with 2% BSA in TPBS(phosphate buffered saline with 0.1% Tween 20) for overnight at 4 C. Theblot was incubated with GRP-94 monoclonal antibody (SPA-850, StressGen)diluted 1:3000 for 1 hour at room temperature. Then it was washed withTPBS buffer for another one hour, and incubated with horseradishperoxidase conjugated anti-rat secondary antibody (Sigma, I:4000dilution) for 1 hour. After successive washing with TPBS the membranewas developed with ECL system (Amersham).

A total-protein (9/1 sample) dilution series was blotted and developedparallel with the samples every time, and a calibration curve wascalculated. The changes in the stress protein content was quantifiedusing a Bio-Rad densitometer model 1650) and a Hewlett-PackardIntegrator (11 P 3394A) and corrected according to the calibrationcurve.

Statistical Analysis

Data are reported as means±SEM. Statistical comparison and calculationwas performed by one-way analysis of variance with Posthoc Newman-Keulstest (Pharmacological Calculation System). Statistical significance wasdefined as P<0.05.

3. Results

Significant decrease of relative GRP-94 content is observed in 1 week,2-weeks and 4-weeks diabetic rats. This effect in 1 week and 2-weeksdiabetic animals could completely be reverted uponN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate treatment. Administration of insulin alone or in combinationwith N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride maleate in 1 week and 2-weeks samples almost doubled the levelsof this protein compared to the control state.

In contrast to the previous findings, treatment of rats diabetic for 4weeks by N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride maleate alone, resulted in no significant alteration in therelative amount of GRP-94. Moreover, both in the insulinized groups,irrespective either treated or not byN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate, we observed a recovery of GRP-94 to the control level.

Effect ofN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridinecarboximidoyl chloridemaleate in Protecting Epidermal Cells Against Damages Caused by Exposureto Heat and UV Light 1. Materials & Methods

HaCaT cell line is a spontaneously immortalized, ancuploid humankeratinocyte cell line derived from normal human adult skin (Boukamp etal., J. Cell Biol. 106:761-771 (1988)), HaCaT is a cell line with fullepidermal differentiation capacity, with normal keratinization and withnontumorogenic character. This rapidly multiplying keratinocyte linewith high ditharnol sensitivity is also characterized by the presence ofsteroid receptors. HaCaT cells (4×10⁵ cells per Petri-dish with adiameter of 35 mm) were seeded and grown in DMEM supplemented with 5%fetal calf serum (Gibco, Cat. No. 011-6290H) under a humidifiedatmosphere of 5% CO₂ at 37° C. 24 ours after plating, the cultures wererinsed with PBS and treated by heat or light.

The confluent cultures of the cells were exposed to heat (42, 44, 46,47, 48° C.) or UV light (UVA 1, 2, 4, 6 J/cm²). Heat exposure wasprovided in circulating water baths. As a UV light source, Waldmann PUVA4000 was used with an energy spectrum of the final output between 320 an390 nm, peaking at 365 nm. The energy output was monitored by an IL-1700radiometer equipped with UVA and UVB sensor.

The morphology of the cells was monitored using phase contrastmicroscopy (Opton Axioplan Microscope, with Plan-Apochromat Ph phasecontrast objectives). The following factors were considered asindicators of cytotoxicity: (a) reduced density of adherent cells; (b)loss of regular “cobble-stone” pattern with enlargement of intercellularspaces; (c) alteration of cell shape. e.g., impeded cell spreading,swelling, pycnotic shrinking, fragmentation: and (d) cytoplasmicchanges, e.g., condensation or vacuolization. Cell viability was alsoexamined, using Trypan-blue exclusion test.

2. Results

Sensitivity of HaCaT keranitocytes to heat-stress was determined byexamining their viability. The results indicated that 24 hours afterbeing exposed to heat at a temperature of 48° C., there were practicallyno living cells (2.7%), compared to the control (100%) at 37° C.Viability of HaCaT keratinocytes 24 hrs after a 45° C. heat exposureproved to be 59%. There were no morphological changes in HaCaT cultureshr after the heat exposure. 24 hrs after the heat treatment at 46° C. orhigher temperature significant (p<0.01) cell detachment andmorphological changes occurred, such as the loss of regular“cobble-stone” pattern with the enlargement of intercellular spaces,swelling, pycnotic shrinking, and vacuolization of HaCaT keranitocytes.Following UV exposure, it was found that reduction of viable cells weredirectly correlated with the dose of UVA.

Preconditioning the cells with heat (42° C. for 1 hour) orN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate (at a concentration of 5×10⁵ M for one hour) provided the cellswith protection against a 48° C. heat exposure. When cells were examined24 hours after the 48° C. treatment, it was found that compared to cellsthat were not treated, the cell viability increased to 48% (whenpreconditioned with 42° C.) and 84% (withN-[2-hydroxy-3-(1-piperidinyl)propoxy)-3-pyridinecarboximidoyl chloridemaleate pre-treatment). The most pronounced protection (increase of 140%in viability) could be observed in case of a combined treatment (with42° C. andN-[2-hydroxy-3-(1-piperidiny[)propoxy)-3-pyridinecarboximidoyl chloridemaleate).

Heat preconditioning with 42° C., but not with 44° C. and 45° C.,induced a prominent reduction of cytotoxicity due to UV-light. Atreatment with 42° C. provided protection both at exposure of 2 and 4J/cm². Viability of pretreated HaCaT kerationocytes increased to 132%(at 2 J/cm²) and to 218% (at 4 J/cm²) compared to UV-exposed cellswithout pretreatment (100%).

Effect ofN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridinecarboximidoyl chloridemaleate in Inducing Molecular Chaperon Expression in Human Skin Tissues

Ultraviolet UVB light (290-320 nm) is one of the components of sunlightand is known to cause damage to the skin. The role ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate in reducing damages caused to the skin tissues by the UVBexposure was investigated, as well as the effect ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate in increasing the expression of molecular chaperon in the skintissues.

1. Materials & Methods

Human skin tissues were grafted onto immunodeficiency (SCID) mice. Theexperimental protocol involved a treatment of one group of the mice withN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate (5.0 mg/kg i.p.) and the other with the solvent NaCl solution(300 μl) for 7 days. On day 8, both groups of mice were exposed to UVBlight (100 mJ/cm²) 24 hours subsequent to the exposure, skin biopsieswere taken for histological (sections stained with hematoxylin andeosin), and immunohistological examinations (indirect immunoflourescencetechnique with monoclonal antibody, mAB).

2. Results

In UV-irradiated mice that was pretreated withN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate, clinical signs of injuries due to UV exposure could not beobserved. In contrast, in one of the untreated animals a pustulousreaction of the transplanted area was found. Furthermore, indirectimmunofluorescence studies using mAB hsp72 showed an intense, linearstaining along the basement membrane zone of human and mouse skin of theN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate treated animals. The same reaction could not be observed in theskin of the untreated animals. In addition, a nuclear type of stainingof granulocytes was present in the pustule (inflammatory skin reaction)of the untreated animal.

Accordingly, administration ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloridemaleate to skin tissues resulted in increased formation of hsp72 in skintissues and provided protection against injuries from UV exposure.

3. Determination of the level of HSP-70 from skin: Western blot analysisof proteins derived from UVB andUVB+N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoylchloride maleate Treated Human Skin Grafted in SCID-Mouse.

Methods:

a. Extraction of Total Soluble Protein from Skin.

All steps were carried out at room temperature. Skin (about 9-35 mg) wascut into tiny pieces and homogenized with frosted-glass pestle inEppendorf tubes for 2 min. in (2 μl/μg skin) of 2 times concentratedLaemmli buffer (65 mM Tris-HCl, pH 6.8: 5% β-mercaptoethanol; 2% SDS;10% glycerol; 0.1% bromophenolblue; all of these materials were Sigmaproducts). Then samples were solubilized for another 60 min. withcontinuos shaking. The homogenates were centrifuged at 10.000 rpm for 10min. prior loading to the gel.

b. Electrophoresis and Immunoblotting [5, 7, 8]

Electrophoresis were carried out on 8% polyacrylamide gel with 10 μl ofsample per lane at constant voltage (50 V) for overnight.

Proteins were either stained with Coomassie Brilliant Blue R-250 ortransferred to Immobilone PVDF membrane (Millipore) at constant current(300 mA) for 3 hours at 4° C. in transfer buffer (10 mM CAPS pH 11.10%methanol).

Non-specific sites of the membrane were blocked with 2% BSA in TPBS(phosphate buffered saline with 0.1% Tween 20) for overnight at 4° C.The blot were incubated with HSP-70 monoclonal antibody (SPA-8dc.,StressGen) diluted 1:1500 for 1 hour at room temperature. Then it waswashed three times with TPBS buffer for another one hour, and incubatedwith horseradish peroxidase conjugated anti-mouse secondary antibody(Sigma, 1:1500 dilution) for 1 hour.

After successive washing (three times) with TPBS, the membrane wasdeveloped with ECL System (Amersham).

Effect ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-(Pyridinecarboximidoyl ChlorideMaleate in Activation of HSP Formation and Protection of OxidativePhosphorylation) 1. Background

It has been shown that exposing Saccharomyces cerevisiae cells to heatshock (5 min. at 42-44° C.) resulted in an impairment of coupling ofoxidative phosphorylation and mitochondrial electron transport system,affecting the ability of the cells to synthesize ATP (Patriarca et al.,Bio-chemistry and Cell Biology, 70:207-214, 1992). However, when thecells were pre-treated withN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate for a short period of time at 37° C. prior to heat shock, itlessened the impairment of the coupling and protected mitochondrial ATPsynthesis. Inhibition of cytoplasmic RNA or protein synthesis duringheat shock appears to prevent this protection of mitochondrial activity.Accordingly, one of the roles of hsp seems to be that of protecting thecoupling of oxidative phosphorylation and mito-chondrial electrontransport system which is disturbed when cells are exposed tophysiological stress, e.g., heat shock.

In this section,N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate is administered to cells prior to being exposed to heat shock,and its effect on protecting coupling of oxidative phosphorylation withmitochondrial transport system against the heat stress is examined.

It was also investigated if the activity of AP-1 and P 1 transcriptionfactors can be modulated byN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate in yeast cells exposed to various stress conditions.

2. Materials & Methods

The experimental protocol for determining the impairment of oxidativephosphorylation and mitochondrial ATP synthesis in Saccharomycescerevisiae is provided in Patriarca et al., Bio-chemistry and CellBiology, 70:207-214, 1992, said reference being fully incorporatedherein by reference.

To S. cerevisiae cells that were maintained at 25° C., varyingconcentration ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate was introduced (concentrations between 10 to 100 μM), cells werethen incubated for one hour at 25° C. in the presence ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate. The temperature was then increased to 42° C., and the oxygraphmeasurements were taken as described in the Patriarca reference.

The cells treated with N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloride maleate were tested for concomitant induction ofhsp genes, using Northern Blotting procedure. The method of extracting,purifying mRNAs from eukaryotic cells, and analyzing the RNA thusobtained using Northern blotting procedure is well known in the art anddescribed in Maniatis, and Maresca, et al. Archives Medial Research 24:247-249 (1993), both of the references being fully incorporated herein.The Northern blotting protocol is also described in Example 7 set forthabove. Hsp 26 and hsp70 DNA sequences were used as probes.

3. Results

3.1

FIG. 4 is the Northern blot analysis of hsp26 mRNA induced in S.cerevisiae, illustrating the results obtained from the experiment. Theconcentration of the chemical compound administered to the cells and theduration of incubation in the compound are indicated.

In the cells that were incubated in the presence ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate (10-100 μM) for 5 min. to 1 hour at 25° C., the induction of hsp26 was observed. The results also seem to indicate that the inductionoccurs even after 5 minute incubation.

It was also found that concentrations between 10 and 100 μM of testcompound was effective in lessening the impairment of the couplingbetween oxidative phosphorylation and mitochondrial electron transportsystem that results from heat shock. The protection from the impairmentof mitochondrial ATP synthesis was in the range obtained whenthermotolerance was induced by pre-conditioning the cells by exposingthem to the intermediate temperature of 37° C. (40-60% protection).

3.2

The effect of benzyl alcohol on (a) adenylate cyclase activity and (b)the physical state of bovine thyroid plasma membranes are shown on FIG.5. Effect of benzyl alcohol on (a) the adenylate cyclase activity and(b) the physical state of bovine thyroid plasma membranes. The change inadenylate cyclase activity (a) is shown for basal (o-o). TSH-stimulated(−), forskolin-stimulated (ε-ε), choleratoxin-stimulated (∇-∇), andfluoride-stimulated (0-0) enzyme activity. The membranes were incubatedwith the drugs prior to the addition of coupling factors. The physicalstate of the membrane was evaluated by following the steady-statefluorescence anisotropy (b) of several fluorophores embedded in themembranes: DPH (o-o). 12-AS (−) and TMA-DPH (ε-ε). Measurements wereperformed at 37° C. The flourophore/lipid molar ratio was always 1:500.

3.3

Our experiments proved thatN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridine carboximidoyl chloridemaleate exerts a significant influence on the activity of AP-1 and itseffect is determined by the actual metabolic conditions of the cells.While N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3 piridinecarboximidoylchloride maleate increases the activity of AP-1 if the supply ofnutrients is inadequate, in rich medium it does decrease the activity ofthe factor. The effect of the drug is most pronounced in dense, late logcultures. The opposite tendency could be seen for P 1. Its activitydecreased if N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloride maleate was administered to cells in a minimalmedium. It is conceivable, that the downregulation of P-1 was elicitedby the very changes in the cells anti-stress machinery caused by theN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate induced AP-1 activation. It is noted, that P-1 responded to alltypes of stresses but its activity was not influenced by the testmaterial. Our important findings, especially with AP-1 could explainmany facets of the in vivo activity of the drug and will be discussed indetails.

The effect ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate (B) on hsp gene expression in tissue culture is shown in FIG. 6.HeLa cells were transfected with a reporter plasmid construct in whichthe promoter of human hsp70 gene was fused to the luciferase reportergene. The effect of heat shock and/orN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate on the hsp promoter was determined by measuring the activity ofluciferase in luminometer and by determining the hsp protein level(expressed from the chromosomal gene) on Western blot.

Samples are:

1: no DNA control; 2: 10 μg transfected DNA at t₀, no heat shock; 3: 10μg transfected DNA at t₀ 60 min. heat shock 24 h later; 4: as earlier+N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridine carboximidoylchloride maleate (B) at to: δ: 10 μg transfected DNA att₀+N-[hydroxyl-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoylchloride maleate (B) at t₀: 6: 10 μg transfected DNA at t₀ 60 min. heatshock 24 h later N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloride maleate (B) added at the time of heat shock; 7:10 μg transfected DNA at to 60 min. heat shock 24 h laterN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate (B) added at 0 time and at the time of heat shock: δ: 10 μgtransfected DNA at t₀ with no heat shock,N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate (B) added at 0 time and 24 h later.

Effect of N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoylchloride maleate in Inducing Molecular Chaperon in Tumor Cells

Nonlethal heat shock increases the sensitivity to lysis mediated by NKcells by 1.5-fold and that heat shock plus treatment withN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridine carboximidoyl chloridemaleate has a synergistic effect on the usability of K562 cells. Thisadditional effect accounted forN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate clearly resulted from the elevated plasma membrane expression ofhsp72, since in vivo antibody blocking studies (using hsp72 specificmonoclonal Ab) revealed a strong inhibition of NK-lysis.

On FIG. 7, the effect ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate (B) on heat shock induced hsp72 levels is shown.N-[2-hydroxy-3-(1 piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate alone did not increase hsp72 levels while the combination ofheat shock andN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate treatment resulted in a significant increase of hsp72 expressioncompared to heat shock alone.

1. Background

Heat shock proteins are known to be located in the cytoplasm, where theyperform a variety of chaperoning functions. In tumor cells, however, hspis reported to be expressed also on the surface of cell membrane(Ferrarini, M. et al. Int. J. Cancer, 51,613 619, (1992)). Experimentsseem to indicate that increased hsp (e.g. hsp70) on cell surface isinduced by exposure of tumor cells to nonlethal heat shock, and thisincrease correlates with an increased sensitivity of IL-2 specific, CD-3natural killer cells (NK) toward tumor cells. Since NK cells arereported to participate in infiltrating and killing tumor cells in vivo(Kurosawa, S. et al. Eur. J. Immunol. 23:1029, (1993)), this increasedsensitivity of NK cells towards tumor cells allows better targeting ofthe tumor cells by NK cells. Thus, if the expression of hsp can beinduced in tumor cells, with increased hsp on the cell surface, it canallow better targeting and killing of these cells by the NK cells. Inthis section, the effect ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate in inducing expression of hsp72 in tumor cells is examined.

2. Materials & Methods

Human K562 cells, a myeloid tumor cell line derived from a patient withchronic myelogenous leukemia in blast phase (ATCC, CCL243) was used(Lozzio, B C an Lozzio, B B Blood 45:321, 1975). Exponentially growingcells were treated with 5×10⁻⁵ MN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate during the nonlethal temperature (42° C.) for 2 hours. Followinga recovery period of 16 hrs at 37° C., cells were tested for the levelof hsp72 content by flow-cytometry (Multhoff et al., Int. J. Cancer: 61,272-279, (1995)). Treatment withN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate resulted in an enhanced level of hsp72 in the tumor cells.

Interaction of N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloride maleate with Lipid Membranes.A Monolayer Study 1. Background

The mechanism(s) by which stress (physical, pathophysiological, etc.) isdetected as a signal and transduced to the transcriptional apparatus ishitherto unknown. It was assumed that the physical state of the membranelipid matrix, which determines the structure and function of themembrane-bound proteins, is directly involved in the perception oftemperature changes and that under heat shock (HS) conditionsperturbance of membrane structure causes transduction of a signal thatinduces transcription of HS genes. Parallelly with the induction ofstress tolerance,N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate was shown to enhance the efficacy of cells to detect and signalvarious stress conditions by upregulating the expression of somechaperone genes.

It is known that monolayer technique using monomolecular lipid layersspread at the air-water interface is an effective tool for theverification of the presence of interactions between membranes andmembrane active agents. The behavior of the bilayer system is verysimilar to that of the respective monolayer system in many aspects(molecular area of membrane constituents, phospholipase action,orientation of inserted proteins, etc.). By measuring the surfacepressure changes caused by molecules inserted into monolayers it becomespossible to get insight into the molecular dimension of the interaction.

The crucial prerequisite of the assumption that someN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate mediated early triggering events in stress responses may occurin the cell membrane is to serve evidences on the direct physicalinteraction ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate with membrane constituents. The aim of the present study was toinvestigate the interaction ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate with membranes by using different lipid monolayers as modelsystem for biological membranes.

2. Materials and Methods

Monolayer experiments were carried out in a Teflon dish, with a volumeof 6.5 ml, and a surface area of 8.8 cm² at 25° C. in a KSV3000Langmuir-Blodgett instrument (KSV Instruments Ltd., Helsinki, Finland)essentially as described. Monomolecular lipid layers consisting of1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), egg yolkphosphatidylglycerol (EggpG) or bovine heart cardiolipin (BHCL) werespread from chloroform lipid solutions to give the desired initialsurface pressure on a subphase of 10 mM Na-phosphate (pH 7.0). Thesubphase was continuously stirred with a magnetic bar.N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridine carboximidoyl chloridemaleate, dissolved in H₂O, was added underneath the monolayer through ahole in the Teflon chamber connected to the subphase. The injectedvolumes were always <1% of the total subphase volume. The surfacepressure was measured by the Wilhelmy method using a platinum plate. Thesurface pressure increase data were extracted from the raw data files byusing the LB5000 software of the Langmuir-Blodget measuring system.

3. Results

The interaction ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate with different phospholipids was tested by measuring thedrug-induced surface pressure increase of lipid monolayers spread at theair-water interface (FIG. 8.). Monolayers in the present study have beenformed from DPPC, EggPG and BHCL and increasing amount ofN-[2-hydroxy-3-(1-piperidinyl)'propoxy]-3-piridinecarboximidoyl chloridemaleate was added in two concentrations (10⁻⁶M, 10⁻⁵M) to the subphase.The addition of the drug underneath a lipid monolayer resulted in asurface pressure increase which was dependent on the concentration ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate in the subphase in all cases. There was, however, a prominentdifference in the surface pressure profile of the different lipidmonolayers. In case of zwitterionic DPPC, the pressure changed quicklyafter the injection ofN-[2-hydroxy-3-O-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate, then it stayed at a constant level. By using mono layerscontaining the negatively charged BHCL the surface pressure profileshowed a typical insertion kinetics, that is the pressure increased forabout two minutes after which it reached an equilibrium level. In thepresence of PG, the insertion kinetics of the drug was similar to thatobserved with BHCL, however, after reaching a certain value, thepressure started to decrease. The rate of pressure decrease wasdependent on the concentration of the drug in the subphase. One possibleexplanation for this phenomena is the removal ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate-EggPG complexes from the interface since the decrease ofpressure continued, even after reaching the initial pressure of the purelipid monolayer.

To get further insight into the specific interaction ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate with lipid monolayers the drug induced surface pressure increasewas measured at different initial surface pressure (FIG. 8.) The methodof extrapolation to high initial surface pressure allows the estimationof limiting insertion pressures for the molecule, at which it is nolonger able to insert into the monolayer. The extrapolated limitinginitial surface pressures were 89 mN/m and 39 mN/m for BHCL and forDPPC, respectively. In case of monolayers containing the negativelycharged BHCL, the pressure increases were always higher than those foundfor the zwitterionic DPPC by suggesting the importance of electrostaticinteractions.

Our investigations serves with the first evidence that upon theadministration ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate in a physiologically relevant concentrations, it is able tointeract with lipid membranes, in a head-group specific manner.

In FIG. 8, the interaction ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridine carboximidoyl chloridemaleate (B) and the monomolecular lipid layers is shown. At the arrowsN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate was added to the subphase at the indicated concentrations. InFIG. 9, the surface pressure increase is presented after the injectionof N-[2-hydroxy-3-O-piperidinyl)propoxy]-3-piridinecarboximidoylchloride maleate (B) underneath monolayers of BHCL or DPPC at differentinitial pressures. TheN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate concentration in the subphase was 10 μmol. The linear regressionanalysis of the experimental data resulted in correlation coefficientsof 0.844 and 0.995 for BHCL and DPPC monolayers respectively.

The Protective Effect of N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloride maleate Against CytotoxicCytokines and Cycloheximide 1. Background

The purpose of these studies was to investigate a possible connectionbetween the production of cytokines and the pathophysiological changesN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate seems to be protective against.

Our data suggest thatN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate is a cytoprotective agent for tissue cultured cells treated withcytotoxic cytokines.N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate treatment increased the survival rate of TNF treated WEHI 164(and other mammalian) cells. This effect was concentration dependent,but was not directly proportional to the drug concentration. The extentof protection provided by theN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridine carboximidoyl chloridemaleate treatment was variable from experiment to experiment, though theincreased resistance of the treated cells to cytotoxic cytokines wasclearly a tendency in all experiments. The protection provided byN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridine carboximidoyl chloridemaleate treatment was not very high, however, in a living animal eventhis degree of protection could have been sufficient to moderate orprevent pathophysiological processes.

2. Aim of Study

Serum TNF levels and inducibility of macrophages from STZ diabetic andcontrol animals were measured. LPS-induced serum TNF activities weresignificantly enhanced in STZ-induced diabetic rats (6-18 weeks of age)compared with those of non-diabetic rats, during the first month ofdiabetes.

3. Results

The mean serum TNF concentration (measured by radioimmunoassay) of thediabetic group was significantly higher (480±96 U/ml) than in healthycontrols (345±48 U/ml). (Foss et al. 1992 Braz. J. Med. Biol. Res.25,239 reported similar results in human patients). Within the diabeticgroup, there was no correlation between serum TNF levels and duration ofdiabetes. We have not found biologically active TNF in the sera ofdiabetic animals by the cytotoxicity assay on L929 cells. The differencebetween the RIA and cytotoxicity measurements indicate the presence ofhigh levels of soluble TNF receptor antagonists (a protective,anti-inflammatory molecule), suggesting the involvement of TNF indiabetic complications.

N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate had an unexpected proliferative effect on yeast cells (and ondifferent, cultured, normal, diploid animal or human cells). Theinfluence of N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloride maleate was especially impressive in the presenceof low concentrations of the growth inhibitory antibiotic,cycloheximide. Yeast cell colonies grown in the presence ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate and cycloheximide did not show an increased incidence of geneticchanges (antibiotic resistant mutations), only higher metabolicresistance against the inhibitory effect of cycloheximide on proteinsynthesis.

According to our measurements the effects ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate can be traced down to the increased activity of the AP-1transcription factor, which mediates the effects of both mitogenicfactors and different types of stress. The results also indicate thatthe above test compound and similar compounds influence the detixity ofAP-1 and possibly other transcription factors by maintaining the effectsof growth factors and metabolic stress conditions.

In FIG. 10. LPS induced TNF production in vitro of macrophages isolatedfrom STZ diabetic (1) and normal (2) animals, in FIG. 11, theN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate induced protection of keratinocytes against the growthinhibitory effect of cycloheximide, in FIG. 12, theN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate (B) induced protection of cells (endothelial cells) againsttoxic effects of cycloheximide, in FIG. 13.N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridine carboximidoyl chloridemaleate (B) protection of human cervical HeLa cells against the growthinhibitory effect of the antibiotic cycloheximide, in FIG. 14, theN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate (B) induced protection of heart muscle cells against the growthinhibitory effect of the antibiotic cycloheximide, and in FIG. 15, theeffect of N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoylchloride maleate (B) on P1 transcription factor activity in AB1380 yeastcells are demonstrated. Row 6. represents the mean values. Row 5. isempty. In FIG. 16, the effect ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate on API transcription factor activity in JF1 yeast cells aredemonstrated. Row 11. represents the mean values. In FIG. 17, the effectof N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoylchloride maleate (B) on P1 transcription factor activity in AB1380 yeastcells are demonstrated. Row 6. represents the mean values. Row 5. isempty.

Cardioprotective Effects ofN-[hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate in Isolated Rat Hearts

1.

The objective of the study was to investigate the cardioprotective andantiarrhythmic effect ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate in isolated working rat hearts.

2. Methods

After a 10-min. aerobic working perfusion, hearts (n=10 in each group)were subjected to a 10-min. coronary occlusion followed by a 3-min.reperfusion in the presence of 0.05, 0.5, 5.0, 20.0, and 50 mg/LN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridine carboximidoyl chloridemaleate, respectively.

In the further studies rats were pretreated with the most effective (20mg/kg) dose ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate, one and five hours before isolation of the hearts,respectively. After excision of the hearts, they were subjected to thecoronary occlusion protocol detailed above while perfused inpresence/absence of 20 mg/lN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate.

In separate experiments the effects of heat stress, ischemia,N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridine carboximidoyl chloridemaleate and their combination were studied on myocardial HSP-70 proteincontent. Isolated hearts were subjected to 15 min. heat-stress (42° C.),global normothermic ischemia, andN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridine carboximidoyl chloridemaleate perfusion followed by 120 and 180 min. reperfusion,respectively.

3. Results

Before ischemia,N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate increased coronary flow (CF) with a bell-shapedconcentration-response relationship. Other cardiac functional parameterswere not changed by lower concentrations of the drug.N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate at 50 mg/L caused significant bradycardia, a reduction in aorticflow (AF) and +dP/dt_(max), and an increase in left ventricularend-diastolic pressure (LVEDP) before ischemia. In the control group,coronary occlusion markedly decreased CF, AF, +/−dP/dt_(max), andincreased LVEDP.N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate alleviated ischemia-induced deterioration of cardiac functionwith a bell-shaped concentration-response relationship. Theconcentration of 20 mg/LN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate showed the most pronounced anti-ischemic effect. Reperfusionafter 10 min. coronary occlusion triggered ventricular fibrillation (VF) in all hearts of the control group. Higher concentrations ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate resulted in a dose-dependent antiarrhythmic effect.

After one hour pretreatmentN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridine carboximidoyl chloridemaleate still afforded cardioprotection, and potentiated the acuteeffects of the compound. Five hours after pretreatment, thecardioprotective effect was not observable, however, some of the acuteeffects of N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloride maleate perfusion were increased.

The compound alone did not increase myocardial HSP-70 content.Stetyocardial HSP-70 content was markedly elevated due to heat stress,however, ischemia resulted in a mild HSP-70 elevation. Nevertheless,when ischemia was induced in the presence of 20 mg/1N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate, HSP-70 content was increased to approximately the same level asfound after heat stress.

We conclude thatN-[2-hydroxy-3-O-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate exerts an anti-ischemic, and an antiarrhythmic effect. Theconcentration of 20 mg/L was found to produce both marked anti-ischemicand antiarrhythmic effect in the isolated rat heart. When the directanti-ischemic effect of the drug disappears after one hour, it stillincreases the degree of protection afforded by acuteN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridine carboximidoyl chloridemaleate treatment. N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridinecarboximidoyl chloride maleate and ischemic stress together induces arapid de novo synthesis of HSP-70 in the rat heart.N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate alone does not affect HSP-70 synthesis.

In FIGS. 18-19, the hsp protein levels, determined by Western blottingare demonstrated from control, heat shocked, item treated, theN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate treated (B) andischemia+hydroxy-3-(1-piperidinyl)propoxy]-3-pyridine carboximidoylchloride maleate treated (Ischemia+B) rats followed by 2 (FIG. 18.) or 3hours (FIG. 19) recovery.

Chaperon-Booster N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloride maleate in Prevention andRepair of Skin Damages: Ultraviolet Light B Protection in Human SkinGrafted Severe Combined Immunodeficiency Disease Mice and AcceleratedWound Healing in Diabetic Rat 1. Background

Hsps appear to play a general role in the physiological protection ofthe skin from environmental stress. As molecular chaperones theyparticipate in prevention and repair of damages caused by variousexposures, such as mechanical trauma, light, heat and chemical injuries,infections, etc. (E. V. Maytin, JID 104:448, 1995.) In pathologicalconditions, such as diabetes mellitus attenuated function of certainhsps has been reported (M. Cherian and E. C. Abraham, Biochem. Biophys.Res. Com. 212:184, 1995). SinceN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate was shown to act as a chaperon booster (Vigh et al., inpreparation) we would expect that the drug is able to enhance mostvarious protection and repair mechanisms.

The purpose of this study was to test the effect of systemic (i) andtopical (ii) administration ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate:

(i) in protection against UVB light induced skin injury in human skingrafted on severe combined immunodeficiency disease (SCID) mice,

(ii) in repair of destroyed wound healing process in STZ-diabetic rats.

2. Methods

(i) Human skin transplanted SCUD mice treated byN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate (5.0 mg/kg i.p.) or vehicle were exposed to UVB light (100mJ/cm²). After 24 h skin biopsies were taken for histologicalexamination and for hsp72 determination using immunohistological andWestern blotting techniques. Pretreatment withN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate prevented UVB light induced skin injury determined clinicallyand histologically. Intensive hsp72 staining of linear basement membranecould be observed by immunofluorescence technique and increased amountof hsp72 was measured by Western blotting ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate treated skin samples.

(ii) Streptozotocin-induced diabetic (STZ) rats with partial—to fullthickness thermal wounds created on bilateral thoracic depilated skin byelectroheating probe (3 mm of diameter; 60° C.; for 30, 60 and 90 sec.),treated by topical application of 1%, 2% or 4%N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate containing cream or vehicle were used to determine the woundhealing in form of self-control, side to side comparison. Wound closurewas recorded photographically and using the digital epiluminescencemicroscopic technique. Wound areas were measured by planimetry 48 h and21 days after heat injury. Level of hsp72 of skin biopsy samples wasdetermined using Western blot analysis.

3. Results

Treatment with 4%N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate containing cream significantly (p<0.01) accelerated woundclosure and ele-vated the hsp72 level in skin biopsy samples compared tovehicle control.

Our results lead to the conclusion that the administration ofN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate provides protection against injuries from UV exposure and haspotential therapeutic applications for the clinical treatment ofconditions with defect in wound repair or after surgical intervention.

In FIG. 20, 21, 22 the effect of 1%, 2%, 4%N-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridine carboximidoyl chloridemaleate (B) containing cream are demonstrated on wound healing. On FIG.23, 24, 25 the results are shown according to the grade of wounds.

In FIG. 26, the photographic pictures of untreated andN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate (B) treated wounds are shown.

FIG. 27, shows the hsp72 protein levels of bioxy specimens from controlN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate (B) (1%, 2% and 4%) treated wounds.

In FIG. 28 the immunohistochemical evaluation of hsp72 protein afterN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-piridinecarboximidoyl chloridemaleate (B) treatment is shown.

In FIG. 29, the hsp72 levels fromN-[2-hydroxy-3-(1-piperidinyl)propoxy]-3-pyridine carboximidoyl chloridemaleate (B) treated and untreated (Control) skin samples of SCID miceare demonstrated.

Evaluation of Stimulatory Effect of Hydroxylamine Derivatives, Accordingto the Invention on HSP72 Expression in Cells Exposed to Stresses a.)Cell Culture Conditions

From the applied cells the 3T3 and L-929 mouse fibroblasts were culturedin MEM medium and grew in monolayer, the U-937 human leukemia cells weremaintained in RPMI-1640 medium, in suspension culture, while the HeLahuman epithelial cells were cultured in DMEM medium and in monolayerform. Cell cultures were maintained as described in example 6.2 (a) withthe difference that cells were cultured in the above mentioned culturemediums.

b.) Conditions of the Experiments

Experiments were carried out applying stress before and after the drugtreatment. Stress was provoked by heat, by chemical agent, HgCl₂treatment. The test compounds were applied in the treatments at the10⁻⁵M concentration.

Cytotoxicity studies, which were performed by a 3 days assay, and wereevaluated by MTT (Cytotechnology 11:49-58) indicated that all of thetest compounds had a 50% growth inhibitory effect at a concentrationlarger than 10⁻⁴ M. Consequently, the concentration used in HSP72studies has no significant cytotoxic effect.

1. Experiments with Heat Stress

These experiment were performed on 3T3 and L-929 mouse fibroblast cellsfurthermore on U-937 human leukemia cells.

The experiments on 3T3 cell applying heat stress were performed asdescribed in the 6.2. (c) point of example 6, with the difference thatstress was induced by a 30 min. exposure at 43° C. temperature andtreatment with the test compounds was performed 15 min. before or 100min. after the heat stress.

Immunodetection was carried out using the HSP72 specific SPA 810(StressGene) primary and the horse-radish peroxidase conjugated A9044(Sigma) secondary antibodies. Densitometric evaluation was performed bya LKB Ultrascan XL densitometer.

Results are summarized in Table 2 and Table 3.

TABLE 2 Stimulatory effect of hydroxylamine-derivatives by the inventionon the heat stress induced HSP72 production in 3T3 cells if treatmentprecedes stress exposure. HSP72 level relative to the stress Compoundsexposed control N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridine- +++carboximidoyl-chloride-maleate5,6-dihydro-5-(1-piperidinyl)-methyl-(3-piridyl)-4H-1,2,4- +++oxadiazine3-(3-piridyl)-5-[(1-piperidinyl)-methyl]-5,6-dihydro-6H-1,4,2- ++dioxazine-(Z)-2-butenedioate (1:1)N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-benz-imidoyl- + chloridemonohydrochlorideN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-N′,N′-diethyl-3- +++pyridine-carboximidamide monohydrochloride3-(3-piridyl)-5-diethylaminomethyl-5,6-dihydro-6H-1,4,2- + dioxazinehydrochloride 3-phenyl-5-[(1-piperidinyl)-methyl]-5,6-dihydro-6H-1,4,2-++ dioxazine hydrochloride/R/(+)-N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridine- +++carboximidoyl-chloride-(Z)-2-butenedioate (1:1)(−)N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridine- +++carboximidoyl-chloride-(Z)-2-butenedioate (1:1)N-[2-hydroxy-3-(piperidinyl)-propoxy]-naphtalene-1- +++ carboxamide3-(3-piridyl)-5-t-butylamino-5,6-dihydro-6H-1,4,2-dioxazine +N-(2-hydroxy-3-piperidino-propoxy)-ethylurethane +N-[2-palmitoyloxy-3-(1-piperidinyl)-propoxy]-3-pyridine- +++carboximidamide monohydrochlorideN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-N′-propyl-urea +++N-(3-chloro-phenyl)-N′-[2-hydroxy-3-(1-piperidinyl)- +++ propoxy]-ureaN-(3-piperidino-l-propoxy)-3-pyridine-carboximidoyl- +++ chloridedihydrochloride O-(3-diethylamino-propoxy)-3-pyridine-carboximidoyl- +++chloride hydrochlorideO-(3-piperidino-propyl)-3-nitro-benzhydroximoyl-chloride +++hydrochloride 1-{[3-(t-butylamino)-2-hydroxy-propoxy]-imino}-1-(m- +++trifluoromethyl-phenyl)-ethane acetateN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-benzylurethane 0N′-[2-hydroxy-3-(1-methyl-1-piperidinillm-1-yl)-propoxy]-N-methyl-piridinium-3-carboximidoyl-chloridc diiodide +++N-hexyl-N′-[3-(1-piperidinyl)-propoxy]-urea ++N-cyclohexyl-N′-[2-acetoxy-3-(1-piperidinyl)-propoxy]-urea 0hydrochloride N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-2-nitro- +++benzimidoyl-chloride monohydrochlorideN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-quinoline- +++ carboximidamidedihydrochloride N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-N,N′-diphenyl-benzamidine 0N,N-dimethyl-N′-[2-hydroxy-3-(1-piperidinyl)-propoxy]-N″-phenyl-guanidine 0N,N-dimethyl-N′-phenyl-N″-[3-(1-piperidinyl)-propoxy]- ++ guanidinehydrochloride N-methyl-N-[3-(1-piperidinyl)-propoxy]-benzamide +hydrochloride 5,6-dihydro-3-(4-chloro-phenyl)-5-[N-methyl-piperidinium-++ 1-E1]-methyl-4H-1,2,4-oxadiazine iodideMethyl-{N-[3-(1-piperidinyl)-propoxy]}-3-pyridine- 0 carboximidatemaleate N-methyl-N-[3-(1-piperidinyl)-propoxy]-m-trifluoromethyl- +++benzamidc hydrochloride N-[3-(I-piperidinyl)-propoxy]-N′-tetramethylene-+++ 3-pyridine-carboxamidine hydrochlorideN-[3-(I-piperidinyl)-propoxy]-N-methyl- 0 N′-(n-hexyl)-urea

TABLE 3 Stimulatory effect of hydroxylamine-derivatives by the inventionon the heat stress induced HSP72 production in 3T3 cells if treatmentfollowed stress exposure. HSP72 level relative to the stress Compoundsexposed control N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3- 0pyridine-carboximidoyl-chloride-maleateN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-2-nitro- 0 benzimidoyl-chloridemonohydrochloride 5,6-dihydro-5-(1-piperidinyl)-methyl-3-(3-piridyl)- 04H-1,2,4-oxadiazine O-(3-piperidino-propyl)-3-nitro-benzhydroximoyl- +chloride hydrochloride N-[2-palmitoyloxy-3-(1-piperidinyl)-propoxy]-3- +pyridine-carboximidamide monohydrochlorideN-hexyl-N′-hexyl-N′-2-hydroxy-3-(1-piperidinyl)- +++ propoxy]-ureaN-[2-hydroxy-3-(1-piperidinyl)-propoxy]- ++ naphtalene-1-carboxamideN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-4- ++pyridine-carboximidoyl-chloride-(Z)- butenedioate (1:1)N′-[2-hydroxy-3-(1-methyl-1-piperidinium-1-yl)- +++propoxy]-N-methyl-piridinium-3-carboximidoyl- chloride diiodideN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3- +++ quinoline-carboximidamidedihydrochloride

In the Table 0 indicates that the treatment altered by ±20% the stressinduced hsp72 level in the cells, while +, ++, +++ indicate 21-50%,51-100% and >100% increase in the hsp72 levels, respectively, relativeto the level of the stress exposed control.

Experiments using heat shock on U-937 leukemia and L-929 mousefibroblast cells were carried out similarly as described above, but drugtreatments in these tests always preceded the heat stress. The compoundN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridine-carboximidoylchloride maleate was tested at the 10⁻⁵ M concentration under theseexperimental conditions.

The treatment resulted in a more than 50% increase in the heat stressinduced hsp72 level.

2. Experiments with Stress Induced by Chemical Agent

These experiments were performed on HeLa, human epithelial and U-937,human leukemia cells using HgCl₂ to induce stress response. Drugtreatment was carried out before cells were exposed to the stress. Testcultures were prepared and treatments were performed as in theexperiments with the 3T3 cells. After the drug treatment cells wereincubated at 37° C. for 15 min. than all the cultures except two (nonstressed control) were exposed to 0.5 μg/ml concentration of HgCl₂ andthe incubation was followed. The induced amount of hsp72 was measured 6hours after the exposure to stress. The applied concentration of HgCl₂resulted in 15-30% of maximal hsp72 level.

On HeLa cellsN-[2-hydroxy-3-(1-piperidinyl)-propoxy-benzimidoyl-chloridemonohydrochloride andN-[3-(1-piperidinyl)-propoxy]-3-nitro-benzimidoyl-chloridemonohydrochloride increased by more than 20% and by more than 50%,respectively the stress induced hsp72 level.

On U-937 cellsN-[2-hydroxy-3-(1-piperidinyl)-propoxy-3-pyridine-carboximidoyl-chloridemaleate treatment enhanced by more than 20% the stress induced hsp72level relative to the stress exposed control.

3. Experiments on Primary Tissue Explants

These experiments were performed on rat spleen and testicular tissueexplants applying heat stress after treatment with the experimentalcompounds. Experiments on spleen suspension were carried out as follows.

Spleens of CFY rats weighing 200 g were removed aseptically, and werehomogenized in 10% fetal bovine serum containing MEM culture medium. Theconcentration of the cell suspension was adjusted to 50-100 mg/5 ml.

Five ml cell suspension was given into each of the culture dishes of 6cm of diameter and the explants were incubated for one hour in a 5% CO₂containing humidified air at 37° C. then cultures were treated with the10⁻⁵ M concentration of the test compounds. After further 15 min.incubation at 37° C. the cultures were exposed to heat shock at 43° C.for 30 min. in an incubator. The amount of the induced hsp72 wasmeasured after a further 6 hours incubation at 37° C.

Results are presented in Table 4, and enhanced levels of hsp72 arescored by the same scale as in Table 2, and 3.

TABLE 4 Stimulatory effect of hydroxylamine-derivatives by the inventionon the heat stress induced hsp72 expression in rat spleen explant Levelof hsp72 relative to the Compounds stress exposed controlN-{3-[(1,1-dimethyl-ethyl)-amino]-2-hydroxy- +propoxy}-3-trifluoromethyl- benzamideN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-N′- +++ heptyl-ureaN-(3-chloro-phenyl)-N′-[2 hydroxy-3- +++ (1-piperidinyl)-propoxy]-urea5,6-dihydro-5-(1-piperidinyl)-methyl-3- 0(3-piridyl)-4H-1,2,4-oxadiazine

Experiments on rat testicular explant were performed as follows.

Testis of CFY rats weighing of 200 g were removed in sterile conditionsand the released testicular tubuli were suspended in 10% fetal bovineserum containing MEM culture medium in that way that five ml suspensioncontained 50-100 mg tissue. The explants were incubated in 5% CO₂containing humidified air at 37° C. for one hour, then the cultures weretreated with 10⁻⁵ M concentration of the tested compounds. After afurther incubation for 15 min. at 37° C., explants were exposed to heatshock at 43° C. for 30 min. The amount of the induced hsp72 was measuredafter a further 6 hours incubation at 37° C.

Results are summarized in Table 5, and the increase in hsp72 level isscored by the same scale as in Table 4.

TABLE 5 Stimulatory effect of hydroxylamine-derivatives by the inventionon the heat stress induced hsp72 expression in rat testicular explantLevel of hsp72 relative to the stress Compounds exposed controlN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridine- +++carboximidoyl-chloride maleate5,6-dihydro-5-(1-piperidinyl)-methyl-3-(3-piridyl)-4H-1,2,4- ++oxadiazine N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-benzimidoyl- +++chloride monohydrochlorideN-{3-[(1,1-dimethyl-ethyl)-amino]-2-hydroxy-propoxy}-3- 0trifluoromethyl-benzamideN-[2-benzyloxy-3-(1-piperidinyl)-propoxy]-3-pyridine- ++carboximidoyl-chloride-(Z)-2-butenedioate (1:1)3-(3-piridyl)-5-diethylaminomethyl-5,6-dihydro-6H-1,4,2- ++ dioxazinehydrochloride N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-4-acetamido- 0benzamidine monohydrochloride3-(3-piridyl)-5-t-butylamino-5,6-dihydro-6H-1,4,2-dioxazine 0N-[2-palmitoyloxy-3-(1-piperidinyl)-propoxy]-3-pyridine- 0carboximidamide monohydrochlorideN-hexyl-N′-[2-hydroxy-3-(1-piperidinyl)-propoxy]-urea 0N-(3-piperidino-1-propoxy)-3-pyridine-carboximidoyl-chloride ++dihydrochlorideO-(3-diethylamino-propoxy)-3-pyridine-carboximidoyl-chloride 0hydrochloride O-(3-piperidino-propyl)-3-nitro-benzhydroximoyl-chloride++ hydrochloride 1-{[3-(t-butylamino)-2-hydroxy-propoxy]-imino}- +1-(m-trifluoromethyl-phenyl)-ethane acetateN-{3-[1,1-dimethyl-ethyl)-amino]-2-hydroxy-propoxy}-3- +trifluoromethyl-benzimidoyl-chloride monohydrochlorideN-[3-(diethylamine)-2-hydroxy-propoxy]-3-trifluoromethyl- +benzimidoyl-chloride monohydrochlorideN-[2-palmitoyloxy-3-(1-piperidinyl)-propoxy]-3-pyridine- +++carboximidoyl-chloride dihydrochlorideN-hexyl-N′-[3-(1-piperidinyl)-propoxy]-urea 0N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-nitro- + benzimidoyl-chloridemonohydrochlorideN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-2-nitro-benzimidoyl- ++ chloridemonohydrochloride N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-N′-heptyl-urea++ N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-1-isoquinoline- +++carboximidamide dihydrochlorideN-methyl-N-[3-(1-piperidinyl)-propoxy]-benzamide + hydrochloride5,6-dihydro-3-(4-chloro-phenyl)-5-[N-methyl-piperidinium-1- +yl]-methyl-4H-1,2,4-oxadiazine iodideN-[3-(1-piperidinyl)-propoxy]-tiophene-2-carboximidoyl- ++chloride-hydrochloride

Measurement of the level of HSP mRNA in the thoracic aorta of Rats withGenetic Hypertension

Rats with genetic hypertension were divided into four groups of four.The groups were treated daily, orally, the first group withphysiological saline, the second group withN-[2-hydroxy-3-(1-piperidinyl)]-propoxy-3-pyridine-carboximidoyl-chloridemaleate (20 mg/kg) for 8 days, the third group withN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-benzimidoyl-chloridemonohydrochloride (5 mg/kg) for 20 days and the fourth group withN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-2-tiophene-carboximidoyl-chloridemonohydrochloride (5 mg/kg) for 20 days. Animals were sacrificed, aortaswere isolated, snap frozen in liquid nitrogen and were stored at −70° C.till they were used.

a) Morphological Examination of the Thoracic Aorta

The examination was performed according to published methods (Br. J. ofPharmacol., 1995; 115, 515-420). A 1 mm² area of the thoracic aorta wasexcised and fixed in 2.5% glutaral-dehyde at room temperature. Postfixation was performed in 1% osmium-tetroxide for one hour. The tissuewas dehydrated in ethanol and was embedded in Durcupan ACM. Pictureswere taken by a Hitachi 7100 electronmicroscope and were evaluatedqualitatively.

It was observed that theN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridine-carboximidoyl-chloridemaleate and N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-benzimidoyl-chloridemonohydrochloride andN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-2-tiophenecarboximidoyl-chloridemonohydrochloride treatment facilitated the regeneration of the cells ofaorta on an average and strong fashion, respectively.

b) Quantitative Measurement of hsp70

Experiments were carried out by the quantitative reverse transcriptionpolymerase chain reaction. The principle of the method is that if twovery similar, but distinguishable templates are amplified in the samePCR reaction then the ratio of the their products is not changed duringthe process. When the initial amount of one of the templates is knownand the relative amount of the products is measurable then the amount ofthe unknown initial template can be calculated. In the most frequentlyused method the known template (competitor) and the unknown template(target) differ only in the length, the competitor is shorter,consequently the PCR products are separable based on their size.

RNA was isolated from the tissues by a guanidinium-isocyanate method(Chomczynski P. and Sacci N.: Anal Biochem. 162: 156, 1987). Theconcentration and the quality of the nucleic acid were evaluated byspectrophotometer and by agarose gel electrophoresis in denaturingcondition (Shambrook J. et al.: Molecular Cloning. A Laboratory Manual,Cold Spring Harbour Laboratory Press, 1989). The isolated RNA was storedat −70° C.

A fragment was constructed from a hsp-70 gene coding cDNA by splicingout an internal sequence (PCR-splicing, Riedy M C et al.: Biotechniques18: 70, 1995). The fragment was amplified by primers specifically bindto the external part of the construct (Erlich A.: PCR Technology,Principles and Applications for DNA Amplification. Stockton Press, 1989)and after measuring the concentration of the product it was stored at−70° C.

The reverse transcription was performed by standard conditions using 1μg isolated RNA/sample with the help oligo dT (dTI6) primer. (ShambrookJ. et al., the same as above)

Equal amount of cDNAs, prepared from RNA samples were mixed with variousamounts of synthetic competitor (derived from 3, 10 times serialdilutions) and the templates were amplified by polymerase chainreaction. The temperatures applied during the cycles were thefollowings: denaturation (95° C., 1 min.), annealing (58° C. 1 min.),synthesis (72° C., 0.5 min.).

After PCR amplification the products were separated on agarose gel (1%)and were stained by ethidium-bromide under standard conditions.(Sambrook et al., the same as above). The stained DNA fragments werevisualized by UV-transluminator for photography. The amount of the PCRproducts was measured by densitometry from the negatives of the photos.

It was observed that the hsp-70 level in the thoracic aorta of theN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-pyridine-carboximidoyl-chloridemaleate, N-[2-hydroxy-3-(1-piperidinyl)propoxy]-benzimidoyl-chloridemonohydrochloride andN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-2-tiophene-carboximidoyl-chloride-monohydrochloridetreated rats was more than 50% higher than the hsp-70 in the controlanimals.

Examination of the Inhibitory Effect on the Aging of Guinea Pig Skin

The inhibitory effect of the compounds, according to the invention, onthe aging of the skin was examined in guinea pig. The skin of fiveanimals per group was depilated and an 1 cm² area was irradiated from aUV-B source of 100 mJ/cm² of intensity on both sides. After theirradiation one side of the skin was treated with a cream composedaccording to the example 10 and containing 5% (w/w) ofN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-3-pyridine-carboximidoyl-chloridemaleate or N-[2-hydroxy-3-(1-piperidinyl)-propoxy]-benzimidoyl-chloridemonohydrochloride orN-[2-hydroxy-3-(1-piperidinyl)-propoxy]-2-nitro-benzimidoyl-chloride-monohydrochloridewhile the other side of the animals was treated with the same creamwithout the active ingredient. This was a self-controlled experiment.

The treatment started immediately after the irradiation and wasperformed twice daily for two weeks.

The UV-B irradiation resulted in a severe skin injury (vesicle, bull,injury of the epithelium, and wound formation) which healed 4 daysearlier and the size of the wound was significantly smaller if theanimals were treated with compounds according to the invention. Thecompounds facilitated the formation of the epithelium.

The experiment shows that the treatment increased the resistance of theskin against the UV-B irradiation and improved the regeneration of theskin.

Chemical Structures of compounds referenced in specification:

1. A method of increasing expression of a molecular chaperon by aneukaryotic cell comprising: treating an eukaryotic cell of a livingmammalian organism that is exposed to a physiological stressaccompanying allergic diseases, immune diseases, autoimmune diseases,diseases of viral or bacterial origin, tumorous, skin and/or mucousdiseases, epithelial disease of renal tubulus, atherosclerosis,coronarial disease, pulmonary hypertonia, cerebrovascular ischemia,stroke, or traumatic head injury with an effective amount of a chemicalcompound to increase the expression of the molecular chaperon by thecell beyond the amount induced by the physiological stress, wherein thechemical compound is one or more of a hydroxylamine derivativerepresented by formula (I″),

or a salt thereof or any optically active streoisomer thereof, whereinR″ is alkyl or substituted alkyl, A is unsubstituted or substituted arylor heteroaryl, and R¹ is H, unsubstituted or substituted straight orbranched alkyl, cycloalkyl, aralkyl, or aralkyl substituted in the alkyland/or aryl moiety.
 2. The method according to claim 1 wherein the cellis treated before the physiological stress.
 3. The method according toclaim 1 wherein the cell is treated after the physiological stress. 4.The method of claim 1 wherein the cell is a neuronal cell, muscle cell,vessel wall cell, epithelial cell or a cell of the immune system.
 5. Themethod of claim 1 wherein the physiological stress is metabolic,oxidative or local mechanical stress or a stress caused by hypoxia, heatshock, radiation or toxic materials.
 6. The method of claim 1 whereinthe physiological stress causes an increase of reactive free radicals ora cytokine present in the area surrounding the cell.
 7. The method ofclaim 1 wherein one or more of the skin or mucosal disease is caused bydermatosis or ulcerous disease of the gastrointestinal system provokedby physiological stress.
 8. The method of claim 1 wherein the molecularchaperon is a heat shock protein (hsp).
 9. (canceled)
 10. The method ofclaim 1, wherein R″ is ω-amino-alkyl which may be substituted on theamino and/or alkyl chain, and wherein the alkyl chain has 1 to 5 carbonatoms.
 11. The method of claim 10 wherein R″ is an ω-amino-alkyl mono-or disubstituted on the amino, and wherein the amino substituent orsubstituents, independently, are one or two straight or branched alkylor cycloalkyl, or the two amino substituents, together with the nitrogenatom attached thereto, form a 3 to 7-membered saturated hetero ring,which may contain additional hetero atoms.
 12. The method of claim 1wherein A is phenyl, phenyl substituted with one or more alkyl, halo,alkoxy, haloalkyl or nitro, or naphtyl or N-containing heteroaryl whichmay be condensed with a benzene ring, or an S-containing or O-containingheteroaryl.
 13. A method of increasing activity of a molecular chaperonin an eukaryotic cell of a living mammalian organism that is exposed toa physiological stress comprising: treating the cell that is exposed toa physiological stress accompanying allergic diseases, immune diseases,autoimmune diseases, diseases of viral or bacterial origin, tumorous,skin and/or mucous diseases, epithelial disease of renal tubulus,atherosclerosis, coronarial disease, pulmonary hypertonia,cerebrovascular ischemia, stroke, or traumatic head injury with aneffective amount of a chemical compound to increase the activity of themolecular chaperon in the cell beyond the amount induced by thephysiological stress, wherein the chemical compound is one or more of ahydroxylamine derivative represented by formula (I″),

or a salt thereof or an optically active stereoisomer thereof, whereinR″ is alkyl or substituted alkyl, A is unsubstituted or substituted arylor heteroaryl, and R¹ is H, unsubstituted or substituted straight orbranched alkyl, cycloalkyl, aralkyl, or aralkyl substituted in the alkyland/or aryl moiety.
 14. The method of claim 13, wherein the mammaliancell is a human cell.
 15. The method of claim 13 wherein thephysiological stress is metabolic, oxidative or local mechanical stressor a stress caused by hypoxia, heat shock, radiation or toxic materials.16. The method of claim 13 wherein the physiological stress causes anincrease of reactive free radicals or a cytokine present in the areasurrounding the cell.
 17. The method of claim 13 wherein one or more ofthe skin or mucosal disease is caused by dermatosis or ulcerous diseaseof the gastrointestinal system provoked by physiological stress.
 18. Themethod of claim 13 wherein the molecular chaperon is a heat shockprotein (hsp). 19-22. (canceled)
 23. Hydroxylamine derivatives of theformula (I″),

wherein A is phenyl or phenyl substituted with halo or nitro or anN-containing heteroaryl group, R′ is H and R″ is ω-aminoalkyl which ismono- or disubstituted on the amino group, wherein the alkyl chain has 1to 5 carbon atoms and the amino substituent or substituents may beindependently one or two straight or branched alkyl or cycloalkyl, orthe two amino substituents, when taken together with the N-atom attachedthereto, form a 3 to 7-membered heterocyclic ring, or the N—C₁₋₄alkyl-quaternary derivative or the N-oxide thereof, with the proviso,that when A is 3-pyridyl, R″ is other than 1-piperidinyl-methyl.
 24. Thehydroxylamine derivatives of claim 23 wherein A is pyridyl. 25.Pharmaceutical composition, and said composition's pharmaceuticallyacceptable carriers and auxiliaries, for the treatment ofcardiovascular, vascular, cerebral, allergic, immune, autoimmunediseases, diseases caused by viral or bacterial infections, tumorous,skin or mucosal diseases, wherein composition contains 0.5 to 99.5% byweight of a hydroxylamine compound of claim 23.